CHARACTERIZATION OF CARBOHYDRATE-PROTEIN GELS USING

DSC AND CONFOCAL MICROSCOPY

Hernández-Espinosa N., Salazar-Montoya J.A. y Ramos Ramírez E.G.*

Department of Biotechnology and Bioengineering. CINVESTAV-IPN. Mexico City,

073600. MEXICO.
E-mail: eramos@cinvestav.mx
SUMMARY

During the last decade, it has increased the interest in exploring the gelling of aqueous
solutions with a mix of carbohydrates and proteins, due to the functional properties of these
biopolymers as food additives. The characterization of these mixtures is necessary for the
development of new processing methods due to their ability to formation of structures with
improved functional properties. It has been reported that gliadin (prolamin wheat) is capable
of gelling at alkaline pH and temperature of 50 ˚C. Therefore, this work aimed to study the
formation of gels from low methoxyl pectin (LMP) and gliadin to characterize its thermal
properties and observe its distribution in the gels. Dispersions were prepared to LMP at 1 %
and 0.5 % of gliadin, the latter stained with fast green following the methodology proposed by
Beaulieu, 2001. Gel formation was induced by the addition of CaCl2. Differential Scanning
Calorimetry (DSC) was performed weighing 8 mg of gel and heated from 20-350 °C, with a
ramp 10 °C. Structure of the gels was performed using Confocal Microscopy Laser Leica
equipment (diode type) and software Leica Application. The images were obtained with a 63x
lens. LMP was able to form gels in presence of divalent ions Ca2+, gelling mechanism is
described as "egg box" model. Confocal Microscopy showed gliadin dispersed in the gel with
uniform distribution. Calorimetric studies showed a single peak near to 210 °C and ∆H= 1573
J/mol, having a different behavior with respect to the controls. The results showed that it is
possible to design carbohydrate-protein gels with potential properties of interest, which could
be useful in the industry of light foods enriched with wheat proteins.

Key words: Low Methoxyl Pectin (LMP), Gliadin, Confocal Microscopy, Differential Scanning
Calorimetry (DSC).

INTRODUCTION

The gliadins are a source of protein storage in the wheat gluten and are essential for formation;
they determine its viscous nature. In combination with glutenin, they provide significant
viscoelastic properties to bread dough. The low methoxyl pectins (LMP) are obtain by
modification of high methoxyl pectins (HMP) by controlled desterification reaction commonly
carried out by a chemical method. LMPs have the ability to form gels without the presence of
sugar, being an important advantage in the formulation of low calorie foods [5].
Carbohydrates-proteins complex arise from electrostatic interactions between oppositely
charged macromolecules. These interactions induce the formation of different supramolecular
entities, such as aggregation and complex coacervates [1]. The nature of complex
carbohydrate-protein is determined by factors that affect the entropy of the system, like
structure and molecular weight of the components. Gelation of these macromolecular
complexes generally takes place by formation of new bonds between the two biopolymers,
polycation-polyanion electrostatic interactions and gel formation due to mutual exclusion of
each component [4].
In these systems, favorable interactions occur between anionic polysaccharides and proteins
found below their isoelectric point, which can cause increases the gelation temperature,
melting temperature and gel strength, among others [4].
The concentrated dissolutions of two biopolymers (protein-protein, carbohydrate-protein or
polysaccharide-polysaccharide) may be turbid and gradually separated into two translucent
layers, each of which may contain some of the other component. However, before phase
separation, compounds gels are obtained where the gelling biopolymer constitute a continuous
phase network and the second is limited to form inclusions in the discontinuous phase [3].
Systems formed by various gelling agents can form gels fillers, mixed and complex [6]. In this
research were proposed two techniques, confocal microscopy and differential scanning
calorimetry for the characterization of carbohydrate-protein gels.

METHODOLOGY

LMP dispersions gliadin 1% and 0.5% were prepared, the latter with Fast Green FCF ™
stained adapting the methodology proposed by Beaulieu [2]. Gel formation was induced by the
addition of CaCl2. The analysis by Differential Scanning Calorimetry (DSC) was performed
weighing 8 mg gel, heated from 20 to 350 °C, with a ramp 10 °C. The structure of the gels was
analyzed by using a computer Confocal Microscopy (Leica TCS laser (diode type) and Leica
Application Software Ver. 2.4.1). The images were obtained with a 63x lens and scanned
between 640 and 700 nm.

RESULTS

Confocal Microscopy
The gels were prepared with varying thickness (≤ 140 microns) by placing them between a
slide and coverslip, after obtaining the serial sections, for better structure observation of the
gels and take gel micrographs. Using of Leica Application Software (Ver. 2.4.1) was possible
to analyze the structure of experimental gels (LMP-gliadin).
Figures 1 to 4 show the front and side face of a gel obtained by LMP and gliadin. Red dots
(Figures 2 and 4) represent the fluorescence emitted by the chromophore bound to the protein;
can also be seen that the protein is embedded in the three-dimensional gel network and
distributed homogeneously (Figures 1 and 3). Figures in black and white (Figures 2 and 4) are
a repeat of his counterpart in another format of design and red dots to gliadin immersed in the
gel formed by the carbohydrate (black dots).

DSC (Differential Scanning Calorimetry).

As a result of the comparative determination of the thermal properties of carbohydrate-protein
gel and individual components (LMP and gliadin), in Figure 5 is showing the thermogram
corresponding gel LMP-gliadin, which showed a different behavior of the presented controls
(data not shown).The main peak suggests that it is a homogeneous mixture obtained under
suitable processing conditions. The thermal peak was obtained near 210 °C and it was
required a change energy (∆H= 1573 J/g).
1) 2)

Figures 1 and 2. Front view of LMP-gliadin gel.

3) 4)

Figures 3 and 4. Side view of LMP-gliadin gel.

Figure 5. Thermogram corresponding to LMP-gliadin gel.

CONCLUSIONS

With the proposed methodology it was achieved to obtain gels containing LMP-gliadin. The
proteins were uniformly distributed in the three-dimensional polysaccharide network (LMP).
The use of a chromophoric compound can permit to observe the morphology of the
carbohydrate-protein gel by Confocal Microscopy. Also by the use of DSC analysis, we can
determinate the changes in the thermal behavior of a mixed gel.

ACKNOWLEDGEMENTS

To CONACYT for the financial support number 211425 to N.H.E. For their technical support to
Biol. M. P. Méndez Castrejón from Biotechnology and Bioengineering Department and to MSc.
I. J. Galván Mendoza from LaNSE -CINVESTAV (Confocal microscopy).

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