Eur Food Res Technol (2001) 212 : 192–197
ORIGINAL PAPER
P. Crowley 7 H. Grau 7 P. O’Connor
R.J. FitzGerald 7 E.K. Arendt
Effect of glutamine peptide on baking characteristics
of bread using experimental design
Received: 21 February 2000 / Revised version: 28 April 2000
Abstract Glutamine is a conditionally essential amino
acid as a deficit in this residue occurs during periods of
catabolic stress. Glutamine-containing peptides can
therefore be considered as potential functional food in-
gredients. The ubiquitous nature and low cost of bread
makes it a potentially ideal vehicle for the delivery of
glutamine peptide to the body. Glutamine-enriched
peptide products were obtained following enzymatic
hydrolysis of gluten. When glutamine peptide is incor-
porated into bakery products the processing variables
considered optimal for standard bread result in dough
with poor machinability. To optimize the production
process a central composite design was chosen. Water
addition, mix time, rest time and proof time were chos-
en as predictor variables. Each experiment was evalu-
ated based on baking tests, color measurements and
texture analysis. Production processes for standard
bread and bread containing glutamine peptide at 3%
w/w were compared. Mix time was a major influence on
the baking characteristics of bread containing glutam-
ine peptide and standard bread. Slight deviations from
the optimum resulted in substantial increases in mois-
ture loss and decreases in volume. Texture values in
standard bread were strongly influenced by water addi-
tion. Using experimental design a production process
resulting in high quality bread containing glutamine
peptide was developed.
P. Crowley 7 H. Grau 7 E.K. Arendt (Y)
Department of Food Science and Technology,
University College Cork, National University of Ireland
e-mail: e.arendt6ucc.ie
H. Grau
National Food Biotechnology Centre, University College Cork,
National University of Ireland
P. O’Connor
Teagasc, Dairy Products Centre, Moorepark, Fermoy, Co. Cork
R. J. FitzGerald
Department of Life Science, University of Limerick, Ireland
Q Springer-Verlag 2001
Keywords Bread 7 Glutamine peptide 7
Experimental Design 7 Optimization
Abbreviations Glu-pep bread: bread incorporating
glutamine peptide 7 ANOVA: analysis of variance 7
TPA: texture profile analysis 7 kDa: kilodalton
Introduction
Glutamine has numerous important, unique metabolic
functions such as maintenance of nitrogen balance and
stimulation of the immune system. Souba and Smith
have reviewed its physiological significance [1, 2].
These properties suggest that glutamine plays an im-
portant role in health and during critical illness such as
surgery, cancer or burns. Under normal conditions, glu-
tamine is considered non-essential. In the body, it is
synthesized from glutamate due to the action of glu-
tamine synthetase. Amino acid imbalances may result
from strenuous exercise, and consequently induce a
number of phenomena, which are referred to as the
“overtraining” syndrome [3]. During stress, an extra
supply of glutamine from the diet is required. This ex-
tra demand cannot be fulfilled by intake of extra gluta-
mate because the glutamine synthesis capacity is insuf-
ficient. This is the reason why glutamine is called a con-
ditionally essential amino acid [4]. Major problems with
incorporation of the free amino acid into food products
are its poor solubility in water and its instability during
heating. From a physiological point of view it is pre-
ferred to use glutamine bound in a peptide because the
transport systems in the gut preferably process di- and
tripeptides rather than free amino acids [5, 6]. These
limitations can be overcome by using glutamine pep-
tides which are preferentially absorbed by the gut. The
technology for glutamine peptide production is based
on hydrolysis of proteins [7, 8]. Proteins are cleaved
into fragments. By combining ultrafiltration processes
components can be isolated from the protein substrate,
be it milk, vegetable or meat proteins [7]. Presently, ap-
 193

plication developments are growing in the health food, Table 1 Wheat bread formula and process
nutritional and sport areas. It is expected that these in-
Ingredients Quantity (g)
gredients will shift into more regular food products [9].
The ubiquitous nature and low cost of bread makes it a Standard Standard with
potentially ideal vehicle for the delivery of glutamine glutamine peptide
peptide to the body. Glutamine is the most abundant
Flour 1000 1000
amino acid in wheat flour (F35.4 g/100 g). However, in Salt 20 20
terms of being processed by the gut, glutamine peptide Yeast 15 15
products have a better molecular mass distribution pro- Glutamine peptide 30
file than native protein, the vast majority of peptides Water Aa Aa
having a molecular mass of less than 3 kDa. Further-
Parameters Conditions
more, because the glutamine peptide is a water-soluble
protein hydrolysate, its properties have potential to im- Proofer temperature 30 7C
prove baking characteristics. When glutamine peptide Proofer humidity 85%
is simply added to a standard bread recipe, the result- Deck oven temperature 220 7C top, 200 7C bottom
Bake time 30 min
ing dough has poor machinability and produces inferior Mix time Ba
quality bread. To overcome this problem it was neces- Rest time Ca
sary to evaluate the relative contribution of process Proof time Da
variables to the baking characteristics of Glu-pep bread a
Levels for water addition and processing parameters are listed
and standard bread. This was achieved using response in Table 2
surface methodology. A central composite design as de-
scribed by Box and Wilson [10] was selected. This de-
sign greatly diminished the number of experiments re-
quired and provided the means to develop a descrip- culated. A modified texture profile analysis (TPA) was carried
tion of process variable interactions and effects. From out 1.5 h after baking using a TA-XT2i Texture Analyser sup-
porting Texture Expert software version 1.17, both by Stable Mi-
the data obtained through the experimental design, an crosystems Limited, Surrey, England. A 20 mm cylindrical alumi-
optimal procedure was sought for the production of num probe and a 40% compression rate at a test speed of 2 mm/s
high quality Glu-pep bread and standard bread. were used. The measurements were carried out on two 25 mm
slices taken from the centre of the loaf. The measurements were
carried out on three loaves from each batch.

Materials and methods

Experimental design
Commercial bakers wheat flour containing 12.2% protein and
20 mg/kg ascorbic acid was used (Odlum Group, Dublin, Ireland). A response surface methodology study as described by Box and
Dried yeast obtained from Mauri Foods (Camellia, N.S.W., 2142, Wilson [10] was conducted of the relative contribution of predic-
Australia), table salt and potable drinking water were incorpo- tor variables (water addition and processing parameters) to bread
rated in the formula. A glutamine peptide powder obtained from characteristics and to determine the optimum processing parame-
enzymatic hydrolysis of gluten, containing peptide bound gluta- ters. Combinations of the four predictor variables were selected.
mine at 28.92 g/100 g powder was used [6]. The powder contained Levels of the predictor variables were calculated using the formu-
95.7% of total peptides less than 3 kDa and 81.1% of total pep- lae given in Table 2. Levels were determined from preliminary
tides less than 1 kDa. baking tests using normal and extreme values. The complete ex-
perimental design, four factors at three levels, required 3 4p81
formula combinations. Based on a central composite design plan,
Preparation of the bread the number of tests could be reduced to 30 as shown in Table 3.
Response variables chosen to determine bread quality character-
The preparation of dough and bread followed a standardized pan istics for each combination were crust color (Lab* values); bake
bread baking procedure as shown in Table 1. After mixing, the loss and volume yield of the bread; hardness, gumminess and che-
dough was kneaded, divided into ca. 400 g pieces and rested in winess of the crumb.
the proofer. Following rest time each piece was divided into six
67B1 g pieces, molded, pan-proofed and then baked. The six
mini-loaves derived from each dough piece were termed as a
batch. For purposes of comparison, each batch was baked with
and without glutamine peptide. After baking, the bread was al- Table 2 Levels of water addition and processing parameters
lowed to cool for 45 min. All measurements were taken using a
batch as one collective unit. Predictor Name (unit) Level a
variable
P2 P1 0 1 2
Evaluation of the bread
A Water addition (% b) 55.5 57 58.5 60 61.5
CIE Lab* values were measured using a CR300 series Chroma B Mix time (s) 30 45 60 75 90
Meter (Minolta, Osaka, Japan). This system defines color in C Rest time (min) 15 20 25 30 35
terms of three dimensions: lightness (L), green to red (a) and blue D Proof time (min) 30 40 50 60 70
to yellow (b). The bread volume was measured one hour after
a
baking by the rapeseed displacement method. Bake loss (% mois- See Table 3
b
ture loss) and volume yield (ml of product/100 g flour) were cal- Flour Basis
194

Table 3 Central composite design a,b

P2 P1 0 1 2

C D/B P2 P1 0 1 2 P2 P1 0 1 2 P2 P1 0 1 2 P2 P1 0 1 2 P2 P1 0 1 2

P2
P1
P2 0 X
1
2
P2
P1 X X X X
P1 0
1 X X X X
2
P2 X
P1
0 0 X X 6c X X
1
2 X
P2
P1 X X X X
1 0
1 X X X X
2
P2
P1
2 0 X
1
2
a
Apwater addition; Bpmix time; Cprest time; Dpproof time
b
P2, P1, 0, 1 and 2 are the levels of water addition and processing parameters as given in Table 2
c
Center point run was repeated six times to determine reproducibility of the method

Model selection and analysis of the response variables
For design evaluation, a quadratic model was selected. This was
the highest order model that did not have aliased terms. For each
of the responses, model summaries and lack of fit tests were used
to determine the most accurate model. To avoid extrapolation er-
rors quadratic models were chosen for analysis of bake loss and
volume yield of Glu-pep bread. All other models were selected
based on lack of fit tests.
fected by variation of processing parameters. The val-
ues for all other responses were allowed to fall within
the range of maximum and minimum values found in
the design. Optimized processing parameters for Glu-
pep bread, with predicted and actual responses are
shown in Table 5. Optimized processing parameters for
standard bread and predicted responses are also
shown.

Statistical evaluation
Analysis of variance (ANOVA) at 99% confidence level was used
to determine significant influences of predictor variables on the
product characteristics of Glu-Pep bread and standard bread (Ta-
ble 4). Primary and secondary influences on product characteris-
tics by predictor variables were determined using perturbation
plots. These show how each response changes as a factor moves
from medium level, with all other factors held constant at the me-
dium level. Primary and secondary influences are indicated in Ta-
ble 4 where relevant.
Results
Baking tests
To illustrate the effects of adding glutamine peptide to
bread, three-dimensional diagrams for bake loss and
volume yield were generated as a function of the two
more influential predictor variables. The other two var-
iables were held constant at the optimum level (Fig. 1).
The reticulated planes in the three-dimensional dia-
grams (response surfaces) reflect predicted combina-
tions of variables necessary to achieve the response cal-
culated for both standard bread and Glu-pep bread.

Optimization of the production process

Bake loss
Characteristics chosen for optimization in Glu-pep
bread were bake loss (minimum) and volume yield Moisture loss was higher in both standard bread and
(maximum). These characteristics were significantly af- bread containing glutamine peptide than is normally re-
 195

Table 4 Significant influences of predictor variables on bread quality characteristics a

Product Glu-pep bread Standard bread

characteristics
Processing parameter b

WA MT RT PT WA MT RT PT

CIE a* value x x2 x1 x1
Bake Loss x1 x x1 x x2
Volume Yield x x2 x x1 x1
Hardness x2 x1
Gumminess x2 x1
Chewiness x2 x1
a
Results from ANOVA calculations at 99% confidence level.
b
WApwater addition, MTpmix time, RTprest time, PTpproof time

Table 5 Optimized processing parameters and predicted results for bread incorporating glutamine peptide a

Processing parameter Level

Glu-pep bread Standard bread

Water addition (%) 57.7 57.4

Mix time (s) 63 75
Rest time (min) 26 27
Proof time (min) 55 52

Product characteristic Results

Predicted (low/high) b Actual Predicted (low/high) b Actual

Glu-pep bread Standard bread

CIE l* value 47.1 53.0 49.7 53.3 59.8 55.5

CIE a* value 15.0 17.1 16.0 13.7 15.4 14.4
CIE b* value 27.2 31.3 28.9 31.1 34.4 32.1
Bake loss (%) 18.3 21.9 19.4 16.6 21.3 19.1
Volume yield (ml/100 g) 439 535 493 386 489 451
Hardness (g) 349 612 450 410 863 505
Gumminess 233 392 302 274 546 401
Chewiness 231 338 272 289 530 408
a
Optimized processing parameters and predicted results for standard bread are shown for comparison
b
Prediction at 95% confidence level

ported. This was due to the small size of the loaves used
in the study (67B1 g). Lower bake loss was achieved at
low levels of water addition. Bake loss increased as wa-
ter addition increased due to evaporation of increasing
levels of unbound water. Mix time was a less influential
parameter but generally increasing mix time decreased
bake loss. Bake loss in Glu-pep bread varied from
about 19–23% across the design. In the case of standard
bread there was a zone of low bake loss in the medium
ranges of water addition and mix time. High mix time
with low water addition or vice versa gave similarly low
results. Other combinations caused an increase in bake
loss.
time allowed the dough to reach greater volume but
over-proofing ( 1 60 min.) resulted in collapse. Varia-
tion of the parameters did not have such a pronounced
effect on volume yield in standard bread. Again high
proof time and mix time produced the best results.
Crust characteristics
Addition of glutamine peptide produces a significantly
darker (lower CIE L* value), more red (greater CIE a*
value), and less yellow (lower CIE b* value) bread. A
thicker crust was observed in Glu-pep bread.

Volume yield
High proof time and mix time gave the highest and
most desirable result. As with bake loss mix time had a
considerable influence on volume yield. Longer proof
Discussion
This study showed that the properties of wheat dough
and wheat bread are significantly affected by addition
196
Fig. 1 Influences of predictor variables on the baking characteris-
tics of Glu-pep bread and standard bread. (a) Influence of water
addition and mix time on bake loss in Glu-pep bread (constants:
rest time 26 min, proof time 57 min). (b) Influence of proof time
and mix time on bake loss in standard bread (constants: water
addition 57.4%, rest time 27 min). (c) Influence of proof time and
mix time on volume yield in Glu-pep bread (constants: rest time
26 min, water addition 57.7%). (d) Influence of proof time and
mix time on volume yield in standard bread (constants: rest time
27 min, water addition 57.4%)
of glutamine peptide. The design plan chosen was suc-
cessfully used to develop optimal procedures for the
formulation of bread containing glutamine peptide and
standard bread. Because of the large number of re-
sponses, contour overlay methodology was impractical.
Optimization of the bread making procedure was done
using a multiple response method called desirability
[11, 12]. This method incorporates priorities and desires
into the optimization procedure. Using this formula,
Glu-pep bread can be produced with several signifi-
cantly improved baking and texture characteristics
when compared to standard bread. Comparison of the
optimized formulae predicted F10% greater volume
yield in Glu-pep bread. The very soluble glutamine
peptide may complex with the wheat gluten forming a
stronger protein network that improves CO2 retention.
Glu-pep bread was predicted to be F25% softer. Gum-
miness and chewiness values were predicted to be
25–30% lower. These predictions, and the high correla-
tion observed between volume yield and hardness val-
ues may be explained by studying the density of the
crumb matrix. As the volume of the product increases
the crumb structure becomes more dispersed and
therefore more easily compressed. Bake loss was not
significantly affected. Without added ingredients, rela-
tively low levels of protein addition can impair bread
quality [13]. This did not occur with the glutamine pep-
tide, which in fact shows some emulsification proper-
ties. Addition of the powder provides stability to the
texture characteristics of Glu-pep bread. None of the
texture characteristics are significantly affected by var-
iation of any of the processing parameters within the
design space. Increasing mix time improves hydration
of the flour particles, which decreases the amount of
unbound water, until the point where dough with mini-
mum mobility is obtained [14]. This may explain why
increasing mix time reduced bake loss in Glu-pep bread
and standard bread. Increased browning in Glu-pep
bread could be attributable to increased Maillard reac-

tion of peptides with reducing sugars, which would
cause the bread surface to darken more readily in the
oven. The thicker crust observed in Glu-pep bread may
have been due to the high water binding capacity of the
powder, which would retard evaporation, and hence
cooling at the surface during baking. The optimized
procedure produced a product with texture and outer
appearance of a very high standard.
Acknowledgement This Research has been part-funded by grant
aid under the Food Sub-Programme of the Operational Pro-
gramme for Industrial Development, which is administrated by
the Department of Agriculture and Food, and was supported by
national and EU funds.
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