Carbohydrate Polymers 81 (2010) 849–854
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Polysaccharide determination in protein/polysaccharide mixtures for
phase-diagram construction
Jacob K. Agbenorhevi, Vassilis Kontogiorgos
∗
Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate, West Yorkshire, Huddersfield HD1 3DH, UK
article info
Article history:
Received 10 November 2009
Received in revised form 25 March 2010
Accepted 30 March 2010
Available online 3 April 2010
Keywords:
Phenol–sulphuric method
Phase diagrams
Mixed systems
Response surface methodology
abstract
The effect of protein removal from protein–polysaccharide mixtures on the determination of total
sugar concentration for phase-diagram construction was explored by response surface methodology.
Phenol–sulphuric acid method was used to estimate the total sugar concentration whereas phase separa-
tion and microstructure of biopolymers in the mixtures were probed by means of fluorescent microscopy.
All the factors studied (trifluoroacetic acid (TFA) volume, protein concentration, precipitation time) had
significant influence on the polysaccharide recovery from the mixtures. The overall pattern of protein
precipitation with TFA was also found to highly depend on protein–time interactions. Images taken at dif-
ferent mixture concentrations and with two different filter sets revealed a phase-separated system with
the guar gum domains surrounded by a continuous protein phase. Experimental measurements as well as
microscopical evidence suggests that, for greater than 60% polysaccharide recovery (±6% error), the pro-
tein content in the mixtures should be < ∼5.5% (w/v) with less than 0.5 mL TFA combined with more than
30 min rest under the influence of TFA. The results of the present investigation showed that when attempt-
ing to construct phase diagram of mixed biopolymer systems the equilibrium phases must be diluted to
a protein content < ∼5% (w/v) before TFA treatment if the maximum polysaccharide concentration is to
be determined.
Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Phase separated biopolymer systems is an active research area
owning to its applications in food, nutraceutical, and drug industry
(Kasapis, 2008; Tolstoguzov, 2003). A first step to investigating such
systems in the liquid state usually involves construction of phase
diagrams of the constituent biopolymers at the specified conditions
of interest in order to identify their compatibility region. Several
methods can be used to construct a biopolymer phase diagram
depending on the desired accuracy. Methods such as the phase-
volume-ratio method (Polyakov, Grinberg, & Tolstoguzov, 1980;
Schorsch, Clark, Jones, & Norton, 1999) or simple visual observation
of the separated phases (Hemar, Tamehana, Munro, & Singh, 2001;
Thaiudom & Goff, 2003) are frequently used to construct phase dia-
grams of binary biopolymer mixtures. However, the most detailed
phase diagram is obtained when the concentration of biopolymers
in the separated phases is determined analytically. The polysaccha-
ride concentration in the phases can be determined with methods
such as refractometry (Bourriot, Garnier, & Doublier, 1999b), or
flow injection analysis (Kontogiorgos, Tosh, & Wood, 2009b), with
the most popular apparently being the phenol–sulphuric method
∗
Corresponding author. Tel.: +44 1484 472488; fax: +44 1484 472182.
E-mail address: v.kontogiorgos@hud.ac.uk (V. Kontogiorgos).
(Antonov, Dmitrochenko, & Leontiev, 2006; Ercelebi & Ibanoglu,
2007; Kim, Decker, & McClements, 2006; Lazaridou & Biliaderis,
2009; Li et al., 2008a; Perrechil, Braga, & Cunha, 2009; Zhang &
Foegeding, 2003).
The original (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956)as
well as the microplate format of the method (Masuko et al., 2005)
for total sugar determination have been developed for pure sugar,
oligosaccharide or polysaccharide solutions free of interferences.
In mixed biopolymer systems, however, interferences resulting
from the protein component may significantly affect the absorp-
tion measurements. Addition of sulphuric acid to proteins results
in various amounts of ammonium sulphate, sulphur and carbon
dioxide. These products will interfere with the overall chemistry of
the reactions making additionally dubious the use of the calibration
curves. Furthermore, blank correction to alleviate signals that origi-
nate from sources other than the polysaccharide is also problematic
since different blanks are needed as the protein concentration
varies in the mixtures. Therefore, estimation of polysaccharide con-
centration is not a straightforward issue and removal of protein
prior to analysis is required. The aforementioned reasons also cre-
ate difficulties in other methods, such as total hydrolysis of the
polysaccharide followed by quantitative HPLC, making these tech-
niques laborious and difficult to be used in practice.
Response surface methodology (RSM) to achieve optimization
provides an effective way to visualize how the system’s response
0144-8617/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.03.056