Response Surface Methodology to Assay

the Effect of the Addition of Proteins

and Hydrocolloids on the Water Mobility
of Gluten-Free Pasta Formulations

V.J. Larrosa, G. Lorenzo, N.E. Zaritzky, and A.N. Califano

Abbreviations

DSC Differential scanning calorimetry

MDSC Modulated differential scanning calorimetry
Peak G Swelling of the starch amorphous region and a cooperative
mediated melting of starch crystallites
Peak M1 Melting of the most stable crystallites
Peak M2 Melting of the amylose–lipid complexes
RSM Response surface methodology
Tc Conclusion temperature
Tg Glass transition temperature
Tmw Ice peak melting temperature
To Onset temperature
Tp Peak temperature
Xi Coded process variables (G, P, W )
Y Corresponding response variable
β Regression coefficients
ΔHm Latent heat of ice melting

V.J. Larrosa • A.N. Califano

Centro de Investigaci
on y Desarrollo en Criotecnologı́a de Alimentos (CIDCA), Facultad de
Ciencias Exactas, UNLP-CONICET, 47 y 116, La Plata 1900, Argentina
G. Lorenzo (*) • N.E. Zaritzky
Centro de Investigaci
on y Desarrollo en Criotecnologı́a de Alimentos (CIDCA), Facultad de
Ciencias Exactas, UNLP-CONICET, 47 y 116, La Plata 1900, Argentina
Departamento de Ingenierı́a Quı́mica, Facultad de Ingenierı́a, UNLP, La Plata, Argentina
e-mail: lorenzogabriel@gmail.com

© Springer Science+Business Media New York 2015 367

G.F. Gutiérrez-López et al. (eds.), Water Stress in Biological, Chemical,
Pharmaceutical and Food Systems, Food Engineering Series,
DOI 10.1007/978-1-4939-2578-0_30
368 V.J. Larrosa et al.

1 Introduction

In gluten-free pasta formulation (suitable for those suffering from celiac disease),
the influence of each constituent has a major importance on the final product
quality, especially water and hydrocolloid contents used to replace the gluten
matrix. Gluten-free doughs are mixed dispersed systems; the dispersion medium
contains several types of dispersed particles, with two main construction mate-
rials—polysaccharides and proteins. Four levels of structural hierarchy in dispersed
food systems can be distinguished: submolecular, molecular, supermolecular, and
macroscopic. Structural functions of a biopolymer depend upon its place in the
structural hierarchy of the product (Tolstoguzov 2000).
The presence of hydrocolloids and proteins in dough may modify the mobility of
water to interact with starch in the gelatinization process (Blanshard 1987). The
amount of water content and the presence of different components such as sugars,
gums, and proteins affect the extent to which the amorphous regions are plasticized
during gelatinization, modifying the corresponding enthalpy and the peak temper-
ature in a differential scanning calorimetry (DSC) thermogram.
The extent of hindered mobility of water molecules is related to the amount of
the unfrozen water content of the systems and their glass transition temperature (Tg)
and influences the conditions for starch gelatinization. The aim of this chapter is to
use response surface methodology (RSM) to analyze the effect of the addition of
proteins and hydrocolloids on water–starch interaction during gelatinization in
gluten-free doughs using a triangular mixture design through the analysis of
thermograms obtained by modulated differential scanning calorimetry (MDSC).
The amount of unfrozen water, water melting temperature, and glass transition
temperature for each formulation are also studied, and their relationship with dough
composition is determined.

2 Mixture Design for Surface Response Analysis

Mixture designs are a special category of response surface designs that are partic-
ularly useful when the actual amounts of a component (factor) do not matter, but
instead the proportion of the whole made up by each component is taken into
account along with the interdependence of factors, by assuming that the factors
must have a sum equal to a constant value (Cornell 2002). In the present work, a
simplex-centroid augmented design with constraints was chosen to study the effect
of adding gums (0.51–2.52 %), protein (0.68–6.70 %), and water (35.5–39.5 %) to a
dough formulation containing fixed mass fractions of corn starch and corn flour (4:1
ratio, 53.5 %), NaCl (1.1 %), and sunflower oil (2.7 %). The design consisted of
12 runs: four points at the extreme vertices of the feasible quadrangular region
(1,2,3,4), four points at the edge centroids (5,6,7,8), one point at the overall centroid
(9), and three added points (A,B,C) to evenly cover the experimental region
(Fig. 1).
Response Surface Methodology to Assay the Effect of the Addition of Proteins. . . 369

Fig. 1 Simplex-centroid 0.0 6

augmented design; mass 0 0.0
fractions are expressed as
coded variables

Xa
5
0.0 0.0

nth
5

an
)

+l
g
4

(g/
0.0

oc
us
ins
0.1 3
8

t
ote
0

be
4

Pr

a
C 3

ng
0.0
5

um
9
6

s(
0.1
5

g/g
2
A B 0.0

)
0.2 1 7 2 1
0 0.0
0.80 0.85 0.90 0.95
Water (g/g)

Surface response analysis was used to determine the relationship of the unfrozen
water of this composite system:

X
3 2 X
X 3 Y
3
Y¼ βi Xi þ βij Xi X j þ β123 Xi ð1Þ
i¼1 i¼1 j¼iþ1 i¼1

where Y is the corresponding response variable, Xi are the coded process variables
(G, P, W ), and β are the regression coefficients. The same methodology was
followed with the peak temperature of water melting.

3 Effect of Dough Composition on Water Mobility

Thermograms were performed from 50  C to 140  C, at a heating rate of

5  C/min, with a modulation of 1  C and a 60 s period, using a modulated DSC
(model Q100, TA Instruments). For each endotherm, onset (To), peak (Tp), and
conclusion (Tc) temperatures of the gelatinization phenomena, as well as the ice
peak melting temperature (Tmw) and the Tg of each sample were also determined.
For all formulations assayed in this work, thermograms show that the characteristic
Tmw between 5 and 8  C and above 65  C starch gelatinization transition was
noticeable (Fig. 2). The latent heat of ice melting (ΔHm) was obtained by integrat-
ing the melting peak of thermograms; this value was later used to estimate the
frozen water fraction in the food material (Roos 1986).
370 V.J. Larrosa et al.

0.0

-0.2

-0.4
Heat Flow (W/g)

-0.6
-0.25

-0.8 Water melting Starch gelatinization

-0.26

Heat Flow (W/g)

-0.27
-1.0
-0.28
M2
-1.2 -0.29
M1
G
-0.30
60 80 100 120
-1.4 Temperature (°C)

-60 -40 -20 0 20 40 60 80 100 120 140

Temperature (°C)

Fig. 2 Complete DSC thermogram corresponding to sample 9. The inset shows the gelatinization
transitions (G) and (M1) and the melting of the amylose–lipid complexes (M2)

Response surface methodology (RSM) was successfully applied to a complex

system such as gluten-free dough to analyze the effect of each component on the
thermal behavior of the dough and to reveal interactions between those compo-
nents. The behavior of the unfrozen water of the composite systems was modeled
using Eq. (1) resulting in a “saddle”-type surface. This process involves several
interactions between components (Fig. 3a), where the largest influence
corresponded to gums and the interaction between gums and water. Mixture of
solutes and water results in altered properties of both constituents. Hydrophilic
groups cause changes in the structure and mobility of adjacent water, and water
causes changes in reactivity and sometimes in the structure of hydrophilic groups
(Fennema 1996). Ions and ionic groups of organic molecules hinder mobility of
water molecules to a greater degree than other types of solutes. From a conceptual
standpoint, it is useful to think of unfrozen water as water that exists in the vicinity
of solutes and other nonaqueous constituents and which exhibits properties that are
significantly altered from those of bulk water in the same system (Fennema 1996).
The water melting peak temperature ranged from 5  C to 8  C for all gluten-
free doughs assayed; dependence on water, proteins, and hydrocolloids content was
modeled. A saddlelike effect, as can be observed in Fig. 3b, was also shown, and
regression coefficients for Tmw exhibited the same trend as in unfrozen water
content; the negative value of the interaction coefficient suggests the presence of an
antagonistic effect between hydrocolloid concentration and water content.
Response Surface Methodology to Assay the Effect of the Addition of Proteins. . . 371

a 0.25 b -5
unfrozen Tmw
water (g/g) (°C)

0.22 -6

LB +
XG
ums
(g/g -7
0.19 )
Gums

0.03
(g/g doug
6

h)
0.0

0.01
0.00
0

0.00
gh)
0.0 0.02

/g dou
5
0
0.1 0.4

2
/g)

0 9

0.0
0.04
0.3

ins (g
s (g

8
h)
tein

0.1 0.3
5
0.950 ug

Prote
0.06 o
Pro

7
0.915 0.3
g/ gd
0.2
0 0.880 ) 6 r(
0.845 (g/g 0.3 ate
Water 0.08 W
0.810 0.3
5

Fig. 3 Surface response of (a) unfrozen water content (g/g total water) and (b) peak melting
temperature (Tmw) as a function of dough composition (expressed as coded variables)

The glass transition denotes a change from brittle to rubbery behavior at a

temperature “Tg.” Tg depends on molecular characteristics, composition, and com-
patibility of the components in the amorphous matrix (Kalichevsky and Blanshard
1992; Roos and Karel 1991) and is related to the water mobility. Tg was determined
from the first derivative of the thermogram at the inflection point for each sample.
Glass transition temperatures were around 26  C (from 24 to 29  C) for all
tested formulations, and it was similar to those reported wheat flour doughs
(Räsänen et al. 1998; Laaksonen and Roos 2000). This temperature was influenced
by the quantities of water added in the formulations. Clearly, it can be observed that
increasing the amount of water available to freeze in the dough produced a decrease
in Tg (Fig. 4a). This result occurs because the average molecular weight of the
mixture decreases.

4 Influence of Dough Composition on Starch Gelatinization

Temperatures

Gelatinization is a key physicochemical transformation that starch granules

undergo when subjected to heat treatment in the presence of water. Gelatinization
induces a number of changes in starch granules such as loss of order, swelling,
exudation of amylose, improved digestibility, granule disruption, enhanced solu-
bility, and increased viscosity. From DSC studies, gelatinization is resultantly an
endothermic process, although it could involve the following two stages (Stevens
and Elton 1971): (1) cleavage of existing hydrogen bonds (endothermic) due to
372 V.J. Larrosa et al.

-24 78
a b
-25

Temperature peak G (°C)

-26 76
Tg (°C)

-27

-28
74
-29

-30
0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 72
0.20 0.25 0.30 0.35
Frozen water (g/g dough)
Frozen water (g/g dough)

Fig. 4 Relationship between the amount of frozen water content in the gluten-free doughs
(g water/g dough) on (a) the glass transition temperature, Tg ( C), and (b) temperature of
gelatinization peak G determined by DSC

hydration and swelling of amorphous regions which facilitates molecular mobility

and then provokes an irreversible molecular transition and (2) formation of new
bonds (involving water) to give a less ordered structure (exothermic) and dissoci-
ation of double helices and expansion of granules as the polymers hydrate.
When dough was heated in the DSC, a characteristic biphasic endotherm
appeared: Peak G which has been assigned to the swelling of the starch amorphous
region and a cooperative mediated melting of starch crystallites, Peak M1 which
corresponds to the melting of the most stable crystallites, and Peak M2 which is
assigned to the melting of the amylose–lipid complexes. The appearance of peaks
G and M1 demonstrates that there was not enough water available in the system for
the gelatinization process (Fig. 2).
For the starch/water system, it was found that onset temperature (To) reflected
the initiation of this process. Onset temperature was followed by a peak (Tp) and
concluded at Tc. After Tc, all amylopectin double helices were dissociated, although
swollen granule structures were retained until more extensive temperature was been
applied (Tester and Debon 2000). Specifically, for the studied cornstarch/water
mixtures, values of 60.6, 66.9, and 74.0  C were obtained for To, Tp, and Tc,
respectively. The temperature range Tc  To ¼ 13.4  C represents the so-called
gelatinization period. Sandhu et al. (2004) found that the To, Tp, and Tc of corn
starches–water mixture ranged from 60 to 69.3  C, 71.5 to 73.1  C, and 76.5 to
78.0  C, respectively. For starches of five open pollinated corn populations, White
et al. (1990) found the gelatinization period between 8.7 and 16.4  C. For the
formulations of gluten-free doughs assayed in this work, To ranged from 67.1 to
69.2  C, Tp from 75.1 to 78.2  C, and Tc from 81.9 to 88.1  C, and the gelatinization
period varied between 14.0 and 18.9  C.
Peak G temperature increased when the amount of water decreased, maintaining
constant gum concentration, that is, less water was available to penetrate the
granule and mediate in the gelatinization process. Once all available water that
Response Surface Methodology to Assay the Effect of the Addition of Proteins. . . 373

was external to the granule was exhausted, the cooperative plasticization process
was arrested, and further gelatinization depended upon increased levels of molec-
ular mobility and granular swelling that were initiated and enhanced by heat. These
conditions demanded heating to higher temperatures. The obtained results also
showed that for a given water content, the addition of hydrocolloids caused a
significant increase ( p < 0.05) of peak G; this was probably the result of the
starch–hydrocolloid interaction, which produced a more stable structure, and less
water mobility due to gums–water interactions, requiring a higher temperature for
disorganization.
Moreover, when the frozen water content of the dough was progressively
reduced (0.334–0.219 g water/g dough), endotherms shifted to higher temperatures
(peak G from 74.98 to 77.34  C) as less water was available (Fig. 4b). As the
amount of frozen water decreased, water mobility diminished and the water-
diffusion-mediated step of the transition (peak G) required more energy. With
respect to the gelatinization enthalpies, no significant differences (P > 0.05) were
observed among the samples studied.
These results support the hypothesis that a reduced level of solvent plasticiza-
tion, resulting from the addition of nonaqueous solutes to a pure water system,
produces elevation of the gelatinization temperature. The reduced level of solvent
plasticization of the amorphous growth ring regions requires input of a greater
amount of thermal energy before the starch granule swells and begins to gelatinize.

5 Conclusions

The effect of protein and hydrocolloid content on water availability in gluten-free

pasta formulation was studied using differential scanning calorimetry. Predictive
regression models were used to plot mixture response surfaces of unfrozen water
content and melting temperature of water as a function of composition. The
response surface methodology led to a “saddle”-type relationship between the
unfrozen water and the dough composition, showing the complex interactions
between single components. Increasing the amount of water in the dough available
to freeze produced a decrease in glass transition temperatures of the systems,
reflecting the higher mobility of macromolecules present.
A biphasic endotherm was observed in the gelatinization transition for all
formulations, and a significant displacement to higher temperatures of the endo-
therms was observed when hydrocolloid content was increased or water content
decreased as water mobility decreased and the water-diffusion-mediated step of the
transition (peak G) needed more energy to occur.
374 V.J. Larrosa et al.

References

Blanshard JMV (1987) Starch granule structure and function: a physicochemical approach. In:
Galliard T (ed) Starch: properties and potential. Wiley, New York, pp 17–18
Cornell JA (2002) Experiments with mixtures: designs, models, and the analysis of mixture.
Wiley, New York, pp. 22–95 and 132–220
Fennema OR (1996) Water and ice. In: Fennema OR (ed) Food chemistry. Marcel Dekker,
New York, pp 17–94
Kalichevsky MT, Blanshard JMV (1992) A study of the effect of water on the glass transition of
1:1 mixtures of amylopectin, casein and gluten using DSC and DMTA. Carbohydr Polym
19:271–278
Laaksonen T, Roos YH (2000) Thermal, dynamic-mechanical, and dielectric analysis of phase and
state transitions of frozen wheat doughs. J Cereal Sci 32:281–292
Räsänen J, Blanshard JMV, Mitchell JR, Derbyshire W, Autio K (1998) Properties of frozen wheat
doughs at subzero temperatures. J Cereal Sci 28:1–14
Roos YH (1986) Phase transitions and unfreezable water content of carrots, reindeer meat and
white bread studied using differential scanning calorimetry. J Food Sci 51:684–686
Roos YH, Karel M (1991) Water and molecular weight effects on glass transition in amorphous
carbohydrates and carbohydrate solutions. J Food Sci 56:1676–1681
Sandhu KS, Singh N, Kaur M (2004) Characteristics of the different types and their grain fractions:
physicochemical, thermal, morphological, and rheological properties of starches. J Food Eng
64:119–127
Stevens DJ, Elton GAH (1971) Thermal properties of the starch–water system. Part
I. Measurement of heat of gelatinisation by differential scanning calorimetry. Starch-Starke
23:8–11
Tester RF, Debon SJJ (2000) Annealing of starch. A review. Int J Biol Macromol 27:1–12
Tolstoguzov VB (2000) Foods as dispersed systems. Thermodynamic aspects of composition-
property relationships in formulated food. J Therm Anal Calorim 61:397–409
White PJ, Abbas IR, Pollak L, Johnson LJ (1990) Intra and interpopulation variability of thermal
properties in maize starch. Cereal Chem 67:70–73