Structure formation in sugar containing pectin gels - Influence of gel composition and cooling rate on the gelation of non-amidated and amidated low-methoxylated pectin [original]

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Kastner, H., Einhorn-Stoll, U., & Drusch, S. (2017). Structure formation in sugar containing pectin gels -

Influence of gel composition and cooling rate on the gelation of non-amidated and amidated low-

methoxylated pectin. Food Hydrocolloids, 73, 13–20. https://doi.org/10.1016/j.foodhyd.2017.06.023

H. Kastner, U. Einhorn-Stoll, S. Drusch

Structure formation in sugar containing

pectin gels - Influence of gel composition

and cooling rate on the gelation of non-

amidated and amidated low-methoxylated

pectin

Accepted manuscript (Postprint)Journal article |

Structure formation in sugar containing pectin gels - Influence of gel composition and

cooling rate on the gelation of non-amidated and amidated low-methoxylated pectin

H. Kastner*, U. Einhorn-Stoll, S. Drusch

Technische Universität Berlin, Food Technology and Food Material Science, Königin-Luise-Strasse

22, D-14195 Berlin, Germany

* Corresponding author.; e-mail-address: h.kastner@tu-berlin.de

Abstract

Gel structure formation and gel properties of low-methoxyl pectin (LMP) and low-methoxyl amidated

pectin (LMAP) with similar degree of methoxylation have been investigated by oscillatory rheological

measurements. The gelling process was examined in a sugar-acid environment matching the

conditions in jams and jellies. Factors studied included cooling rate, calcium content and pH.

Parameters derived from the rheological measurements comprised the gel point, structuring

velocity, initial and critical structuring temperature, average structuring developing rate and loss

factor (tandend).

The influence of the cooling rate on the gelling process of LMP was moderate and the influence on

the final gel properties was significant, tandend decreased with increasing cooling rate. The calcium

content significantly affected the structuring process of LMAP and the final gel properties. At high

calcium content, the gelling process started at a higher temperature but the resulting gels were less

strong. The pH had a significant but partly opposite effect on the gelation of LMP as well as LMAP.

The differences in gelation behavior between LMP and LMAP can be explained by the lower

number of available blocks of free carboxyl groups in LMAP as well as by the formation of additional

hydrogen bonds through the amide groups.

1 Introduction

Pectin is a polysaccharide and extracted from plant cell walls. It is used in the food industry for its

gelling, thickening, stabilizing, and emulsifying properties. Variations in pectin structure enable this

broad range of commercial applications. Depending on the degree of methoxylated carboxyl groups

(degree of methoxylation, DM) pectins are traditionally classified as high-methoxylated pectins

(HMP) with DM > 50% and low-methoxylated pectins (LMP) with DM < 50%. Chemical de-

esterification of HMP in presence of ammonium ions results in low-methoxylated amidated pectin

(LMAP).

For understanding of the gelling process of pectin it is helpful to review the types of junction zones

in pectin gels. A limited number of hydrophobic interactions may be formed between the methyl

ester groups immediately at the beginning of the cooling process and induce the gelation (Oakenfull

& Scott, 1984). These interactions have a rather low energy, a limited working range of about 2 nm

(Walstra, 2002, Chapter 3) and become weaker with decreasing temperature. Upon further cooling,

at lower temperatures hydrophilic interactions between undissociated carboxyl groups of the

galacturonic acid and/or hydroxyl groups of carboxyl, hydroxyl or amide groups can develop via

hydrogen bonds (Oakenfull & Fenwick, 1977; Oakenfull & Scott, 1984). These bonds are also of low

energy and with 0.2 nm their working range is even smaller than that of the hydrophobic

interactions. That means that the pectin molecules have to come in close contact in order to form a

gel network. This can be achieved by a high soluble solid concentration (> 50%) since the resulting

reduced water activity allows the approach of pectin chains (Evageliou, Richardson, & Morris, 2000;

Kastner et al., 2014; Thakur, Singh, Handa, & Rao, 1997). The influence of the hydrogen bonds

gains more importance upon temperature reduction and supports inter-chain association during

network formation. These two types of junction zone formation occur during gelation in all types of

pectin and are typical for a cold-set gelation (Burey, Bhandari, Howes, & Gidley, 2008). However,

gelation of LMP is governed by ionic interactions between dissociated carboxyl groups, typically via

calcium ion bridges (Thibault & Ralet, 2003) in an ionotropic gelation (Burey et al., 2008). Calcium

bridges represent a third type of junction zone formation and start to form immediately after gel

preparation. They are much stronger than hydrophobic and hydrogen bonds and with about 20 nm

their working range is rather long (Walstra, 2002, Chapter 3). Pectin gels with combined or

dominating ionic junction zones require less soluble solids than HMP gels based on hydrophobic or

hydrogen bonds, and can be formed also in sugar-free systems. The ionotropic gelation can take

place in pectin solutions without heating (Ström & Williams, 2003; Vincent & Williams, 2009) and

can be performed also as isothermal titration at room temperature (Fang et al., 2008). It seems to

be possible, however, that ionic interactions formed at higher temperature tend to be initially not

stable but their stability will increase during cooling (Cárdenas, Goycoolea, & Rinaudo, 2008;

Garnier, Axelos, & Thibault, 1993). In LMAP, junction zones are additionally stabilized by hydrogen

bonds involving the amide group (Alonso-Mougán, Meijide, Jover, Rodrılguez-Núñez, & Vázquez-

Tato, 2002; Black & Smit, 1972; Löfgren, Guillotin, & Hermansson, 2006).

Structure formation and properties of pectin gels strongly depend on intrinsic and extrinsic factors

(Endress & Christensen, 2009; Rolin, Chrestensen, Hansen, Staunstrup, & Sørensen, 2009; Yapo &

Gnakri, 2015). Intrinsic factors affecting a system are those related to the composition, e.g. type of

pectin and concentration, agent used for pH adjustment, presence of divalent cations or co-solutes

like sugar. Important extrinsic (technological) factors are e.g. heating and cooling conditions. The

influence of calcium ions depends on the stoichiometric ratio between calcium ions and the

dissociated free carboxyl groups. This ratio is calculated as R = 2[Ca2+]/[COO-] (Axelos & Kolb,

1990; Capel, Nicolai, Durand, Boulenguer, & Langendorff, 2006; Cárdenas et al., 2008; Garnier et

al., 1993; Ström et al., 2007). A theoretical saturation threshold of the R exists at which every

calcium ion in the gel is bound to two dissociated carboxyl group. This threshold is affected on one

hand by the degree of dissociation of the carboxyl groups and, thus, by the pH in the gel system.

The pKa of pectin is about 3.5 (Ralet, Dronnet, Buchholt, & Thibault, 2001), at this pH 50% of the

carboxyl groups are dissociated. On the other hand, the binding of calcium to pectin chains also

depends on the distribution of these groups (block-wise or random). Ionic interactions require a

certain number (blocks) of about 6 to 14 subsequent dissociated carboxyl groups (Liners, Thibault,

& Cutsem, 1992; Luzio & Cameron, 2008; Powell, Morris, Gidley, & Rees, 1982) in order to form

junction zones named as “egg-boxes”. Vincent and Williams (2009) therefore suggested a modified

Reff, in which only dissociated carboxyl groups in blocks are considered. The calculation of their

exact number requires, however, detailed knowledge of the pectin molecular structure. Single

randomly distributed dissociated carboxyl groups may also interact with calcium ions. In case they

are oriented to the outside of the egg-boxes, they could form larger dimer aggregates and even an

extended network (Braccini & Pérez, 2001; Fraeye et al., 2009, 2010). Moreover, excess calcium

ions may be located in the gap between galacturonic acid molecules and interact with other

C-atoms than C6 (Siew, Williams, & Young, 2005), they might course a certain electrostatic

repulsion. The number of rather unspecific or random calcium crosslinks will increase with

increasing calcium ion content. The degree of methoxylation and the distribution of the carboxyl

groups in a more random or more block-wise way can influence the gelling process especially with

respect to the ionotropic gelation (Fraeye et al., 2009; Ngouémazong et al., 2012). According to

Fraye et al. (2009, 2010) pectin with a dominating block-wise distribution of the dissociated carboxyl

groups is able to gel at a lower calcium concentration (R) than pectin with a more random

distribution of free carboxyl groups. At higher calcium content (R>1) pectin gels were found to

become more cross-linked and elastic and a plateau of the storage modulus (G’) was reached only

at very high calcium contents with R up to 5.0. Additional crosslinks might result from random

interactions of calcium ions with single carboxyl groups as described above. When the calcium

content becomes too high, precipitation and/or syneresis can occur and the gel strength may be

reduced (Fraeye et al., 2010; Grosso & Rao, 1998). Gels prepared from amidated pectins were

found to be less sensitive to syneresis (Thakur et al., 1997; Thibault & Ralet, 2003) than those of

non-amidated pectins.

The complex gelling process and gel properties of pectin systems as well as the influence of

different intrinsic and extrinsic factors on the gelation have been successfully examined by a variety

of methods including a wide range of rheology-based methods. In the majority of studies, however,

only one or two intrinsic factors have been varied, like e.g. pectin concentration, co-solutes, ion

concentration, type of ions or acid and alkaline media to adjust the pH (Evageliou et al., 2000;

Fraeye et al., 2010; Gigli, Garnier, & Piazza, 2009; Guillotin, Van Kampen, Boulenguer, Schols, &

Voragen, 2006; Löfgren et al., 2006; Löfgren & Hermansson, 2007; Lopes da Silva & Gonçalves,

1994; Rao & Cooley, 1993; Sousa, Nielsen, Armagan, Larsen, & Sørensen, 2015; Ström et al.,

2007; Tsoga, Richardson, & Morris, 2004). As a consequence, the results of some of these studies

are hardly comparable because of differences in the intrinsic factors of the model system as well as

extrinsic factors in the experimental setup, methodology or measuring equipment. In addition, often

only one parameter, the classical gel point (GP) defined as crossover of storage modulus G’ and

loss modulus G’’ at a certain frequency, was reported (Gigli et al., 2009; Holst, Kjøniksen, Bu,

Sande, & Nyström, 2006; Iglesias & Lozano, 2004). In recent years, the rheological characterization

of a gelling process was significantly improved by using new parameters like the initial structuring

temperature (IST), defined as the temperature at which the first derivation of G’ as a function of time

(dG’/dt) differed from zero for the first time, and the critical structuring temperature (CST) as the

extrapolated temperature of the first strong increase of dG’/dt (Kastner et al., 2014; Kastner,

Einhorn-Stoll, & Senge, 2012a, 2012b; Einhorn-Stoll, Kastner, & Senge, 2012; Einhorn-Stoll,

Kastner, Hecht, Zimathies, & Drusch, 2015). This method allows the evaluation of the gelling

kinetics and the final gel properties and, thus, gives information about systems without clear gel

point. These new parameters are now generally accepted and have been used by several other

groups (Garrido, Lozano, & Genovese, 2015; Sousa et al., 2015; Wang, Hua, Yang, Kang, & Zhang,

2014).

For these reasons, in the last years the overall aim of our group was the investigation of the pectin

gelling process and the final gel properties in a broad study, covering all major factors such as

pectin type, content of calcium ions, pH and cooling rate. Several results have already been

achieved and published and complementary examinations are the subject of the present study. The

kinetics of structure formation of HMP gels, a typical cold-set gelation, as well as the properties of

the final gels were investigated before at varying cooling rates and pH (Kastner et al., 2014). A high

cooling rate promoted early structure formation and resulted in a less elastic gel compared to a low

cooling rate. Varying the pH by differences in the acid concentration showed that an optimum range

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