**1. Introduction**

Polysaccharides and proteins are natural polymers that are widely used as functional ingredients for various food colloids or emulsion formulations. Majority of food emulsions are constituted with polysaccharide and protein combinations. They are the essential ingredients of any food colloid formulation mainly due to their ability to change product shelf life by varying food texture (Schmidt & Smith, 1992; Schorsch, Jones & Norton 1999). Their interaction in the formulation thus finds many applications particularly in new food formulation development. Due to complex formation and creation of nano or micro structures (aggregation and gelation behavior) they generally change the rheological properties of food colloids which may affect the food product texture and colloidal stability (Benichou, 2002; McClements, 2005, 2006, 2007; Dickinson, 2003). Polysaccharide and protein interactions in solution and interfaces have been studied by several groups (Dickinson, 2003, 2008; Bos &Van Vliet 2001; Carrera & Rodríguez Patino 2005; Krägel, Derkatch, & Miller, 2008; Koupantis, & Kiosseoglou, 2009; Mackie, 2009). However, despite the vast advancement made in the recent past, polysaccharide and protein interactions in food hydrocolloids continue to be one of the most challenging topics to understand.

Proteins, being surface active can play major role in the formation and stabilization of emulsions in the presence of polysaccharide, while interacting through electrostatic or hydrophobic-hydrophobic interactions. On the other hand, polysaccharides being hydrophilic in nature generally remain in aqueous phase thus help in controlling the aqueous phase rheology like thickening, gelling and acting as stabilizing agents. The formation and deformation of polysaccharide-protein complexes and their solubility depend on various factors like charge and nature of biopolymers, pH, ionic strength and temperature of the medium and even the presence of surfactant of the medium (Ghosh & Bandyopadhyay, 2011). If pH of the medium is reduced below isoelctric point (p*I*) of the

© 2012 Ghosh and Bandyopadhyay, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

protein present then net positive charge of the protein will become prominent which will interact with negatively charged polysaccharide to form stable electrostatic complex. Similarly, if solution pH increased more than protein p*I*, the net negative charge of protein will tend to form complex with positively charged polysaccharides (Xia & Dubin, 1994; Dickinson, 2008; Turgeon, Schmitt & Sanchez, 2007). Generally, chances of weaker complex formation is more when solution pH is almost equal to protein p*I* , because at that pH range surface charge of protein becomes nearly zero. However, at very high concentration, similarly charged biopolymers repel each other and the net repulsion make the system unstable (separate as two distinct phases) which is known as *thermodynamic incompatibility*. Incompatibility in the system occurs at pH higher than the protein p*I* and at higher ionic strength (Grinberg & Tolstoguzov, 1997). Thus by varying pH and ionic strength of the medium one can achieve a control on the polysaccharide-protein interactions.

Polysaccharides and proteins both contribute to the structural and textural properties of food by changing rheology of food emulsions through their gelling networking system (Dickinson, 1992). Non-covalent interactions between polysaccharide and protein in any emulsion formulation play a major role to change the interfacial behavior and stability of the food colloids. The driving force for these non-covalent interactions is electrostatic interactions, hydrophobic interactions, H-bonding and Van der Waals interactions. Recent literatures also focus on how protein and polysaccharide molecules can be linked together by covalent bond. At pH close to protein p*I* this Maillard-type conjugates were used to improve the colloidal stability and interfacial structure of proteins in certain conditions (Jiménez-Castańo, Villamiel, & López-Fandiňo, 2007; Benichou, Aserin, Lutz & Garti, 2007)). Recent developments in the field describe interfacial physico-chemical properties of polysaccharide-protein mixed systems (Rodríguez Patino & Pilosof 2011). In this chapter, we would like to focus more on polysaccharide and protein non-covalent interaction studies and their effect towards food colloids stability.

## **2. Nature of polysaccharide-protein complex**

Polysaccharide and protein complex formation is mainly driven by various non-covalent interactions, like electrostatic, H-bonding, hydrophobic, and steric interactions (Kruif et al 2001). Protein carries +ve or –ve zeta potential based on the pH of the medium (+ve at pH lower than p*I* and vice-versa). This +ve or –ve electrical charge on the protein chain point towards the presence of different amino acids in the protein molecules and their mode of ionization at different pH ranges (Fig. 1). Carboxylate polysaccharides get deprotonated (become anionic) at a pH range higher than its pKa (Fig. 1). This electrical charge on the back bone of protein or polysaccharide chain is responsible for electrostatic attraction or repulsion between them. Again, presence of -COOH group on the polysaccharide and -NH3, -COOH groups on the protein chain are the sources of hydrogen bonding between these two bio-polymers. Extent of both of this hydrogen bonding and electrostatic interaction depends on the solution parameters such as pH, ionic strength, temperature etc. Except these ionic patches on the bio-polymers, few non-polar segments are also present on the bio-polymers, which are responsible for the hydrophobic staking with each other. Even though solution parameters are important factors to control the different mode of interactions between protein and polysaccharide, type of proteins/polysaccharides, molecular weight, charge density, and hydrophobicity of the bio-polymers are also play significant role towards the extent of complexation between two bio-polymers at a fixed condition.

396 The Complex World of Polysaccharides

protein present then net positive charge of the protein will become prominent which will interact with negatively charged polysaccharide to form stable electrostatic complex. Similarly, if solution pH increased more than protein p*I*, the net negative charge of protein will tend to form complex with positively charged polysaccharides (Xia & Dubin, 1994; Dickinson, 2008; Turgeon, Schmitt & Sanchez, 2007). Generally, chances of weaker complex formation is more when solution pH is almost equal to protein p*I* , because at that pH range surface charge of protein becomes nearly zero. However, at very high concentration, similarly charged biopolymers repel each other and the net repulsion make the system unstable (separate as two distinct phases) which is known as *thermodynamic incompatibility*. Incompatibility in the system occurs at pH higher than the protein p*I* and at higher ionic strength (Grinberg & Tolstoguzov, 1997). Thus by varying pH and ionic strength of the

Polysaccharides and proteins both contribute to the structural and textural properties of food by changing rheology of food emulsions through their gelling networking system (Dickinson, 1992). Non-covalent interactions between polysaccharide and protein in any emulsion formulation play a major role to change the interfacial behavior and stability of the food colloids. The driving force for these non-covalent interactions is electrostatic interactions, hydrophobic interactions, H-bonding and Van der Waals interactions. Recent literatures also focus on how protein and polysaccharide molecules can be linked together by covalent bond. At pH close to protein p*I* this Maillard-type conjugates were used to improve the colloidal stability and interfacial structure of proteins in certain conditions (Jiménez-Castańo, Villamiel, & López-Fandiňo, 2007; Benichou, Aserin, Lutz & Garti, 2007)). Recent developments in the field describe interfacial physico-chemical properties of polysaccharide-protein mixed systems (Rodríguez Patino & Pilosof 2011). In this chapter, we would like to focus more on polysaccharide and protein non-covalent interaction studies

Polysaccharide and protein complex formation is mainly driven by various non-covalent interactions, like electrostatic, H-bonding, hydrophobic, and steric interactions (Kruif et al 2001). Protein carries +ve or –ve zeta potential based on the pH of the medium (+ve at pH lower than p*I* and vice-versa). This +ve or –ve electrical charge on the protein chain point towards the presence of different amino acids in the protein molecules and their mode of ionization at different pH ranges (Fig. 1). Carboxylate polysaccharides get deprotonated (become anionic) at a pH range higher than its pKa (Fig. 1). This electrical charge on the back bone of protein or polysaccharide chain is responsible for electrostatic attraction or repulsion between them. Again, presence of -COOH group on the polysaccharide and -NH3, -COOH groups on the protein chain are the sources of hydrogen bonding between these two bio-polymers. Extent of both of this hydrogen bonding and electrostatic interaction depends on the solution parameters such as pH, ionic strength, temperature etc. Except these ionic patches on the bio-polymers, few non-polar segments are also present on the bio-polymers,

medium one can achieve a control on the polysaccharide-protein interactions.

and their effect towards food colloids stability.

**2. Nature of polysaccharide-protein complex** 

**Figure 1.** Variation of charge density on the polysaccharide and protein chain at various pH ranges.

In general, interactions between proteins and polysaccharides are quite explored where large numbers of report have been published based on the interactions between oppositely charged "protein-polysaccharide" systems (Dmitrochenko et al 1989; Bengoechea et al 2011, Stone & Nickerson 2012). Although electrostatic attraction is the main driving force for the complexation between protein and polysaccharide, but it is also reported that hydrogen bonding and hydrophobic interaction plays a secondary role for stability of the "proteinpolysaccharide" aggregates (McClements, 2006). The extent of hydrogen bonding and hydrophobic interaction also depends on temperature (Weinbreck et al, 2004). In 2009 Nickerson and co-workers(Liu, Low, & Nickerson, 2009) have reported that pea protein and gum acacia complex stabilize at low temperature due to increase in hydrogen bonding interactions and destabilize at high temperature due to decline in hydrogen bonding interactions. Temperature also plays an important role to decide the protein conformations (folded or unfolded). In 2007, Pal (Mitra, Sinha & Pal, 2007) and coworkers have reported that human serum albumin unfolds at higher temperature and undergoes in reversible refolding conformations upon cooling (below 600 c). Unfolded conformations of protein expose more reactive sites (amino acids) to the solvent phase, thus more chances of interactions (or binding) with polysaccharide. Binding of anionic polysaccharides (pH~pKa) to the cationic proteins (at pH<p*I*) result both soluble and insoluble complexes (Magnusson & Nilsson, 2011). Initial binding of polysaccharides (anionic) to the proteins (cationic) cause charge neutralizations, which lead to the formation of insoluble "protein-polysaccharide" aggregates (Schmitt et al, 1998). Further binding of anionic polysaccharides to those neutral aggregates make it effectively anionic, which leads to formation of soluble complexes. But binding of anionic polysaccharides with anionic proteins (pH>p*I*) are also known and governed by the interactions between anionic reactive sites of polysaccharide and small cationic reactive sites of protein (Fig. 2). Binding of anionic polysaccharides to the cationic side of proteins (at pH>p*I*) result in formation of anionic "protein-polysaccharide" aggregates, thus soluble complexes. Therefore, concentration of polysaccharides and pH play an important role towards the solubility of "protein-polysaccharide" aggregates.

Two bio-polymers can exist either in a single phase systems or in a phase separated systems depending on the nature of bio-polymers, their concentration, and solution conditions. When two bio-polymers carry opposite charge, then either they agglomerates to form soluble complexes (single phase) or insoluble precipitates (2-phase system). On the other hand, when two non-interacting bio-polymers mixed together, either they exist in a single phase system (where two separate entities distributes uniformly throughout the medium) or exist as two distinct phases (each phases comprise different bio-polymer). Therefore, in the protein-polysaccharide system, phase separation occurs through two different mechanisms which are *associative phase separation* and *segregative phase separation* (Tolstoguzov, 2006). *Associative phase separation* is the aggregation between two oppositely charged bio-polymers (electrostatic attraction driven), leads to the phase separation, where one phase is enriched with two different bio-polymers (coacervation or precipitation) (Fig. 3). *Segregative phase separation* occurs either due to strong electrostatic repulsion (between two similarly charged bio-polymers) or because of very high steric exclusion (between two neutral bio-polymers). In this case, at low concentration, two biopolymers can co-exist in a single phase whereas at higher concentration, it starts phase separation. (Fig. 3).

**Figure 2.** Interaction between polysaccharide and protein at various pH.

Polysaccharide-Protein Interactions and Their Relevance in Food Colloids 399

398 The Complex World of Polysaccharides

side of proteins (at pH>p*I*) result in formation of anionic "protein-polysaccharide" aggregates, thus soluble complexes. Therefore, concentration of polysaccharides and pH

Two bio-polymers can exist either in a single phase systems or in a phase separated systems depending on the nature of bio-polymers, their concentration, and solution conditions. When two bio-polymers carry opposite charge, then either they agglomerates to form soluble complexes (single phase) or insoluble precipitates (2-phase system). On the other hand, when two non-interacting bio-polymers mixed together, either they exist in a single phase system (where two separate entities distributes uniformly throughout the medium) or exist as two distinct phases (each phases comprise different bio-polymer). Therefore, in the protein-polysaccharide system, phase separation occurs through two different mechanisms which are *associative phase separation* and *segregative phase separation* (Tolstoguzov, 2006). *Associative phase separation* is the aggregation between two oppositely charged bio-polymers (electrostatic attraction driven), leads to the phase separation, where one phase is enriched with two different bio-polymers (coacervation or precipitation) (Fig. 3). *Segregative phase separation* occurs either due to strong electrostatic repulsion (between two similarly charged bio-polymers) or because of very high steric exclusion (between two neutral bio-polymers). In this case, at low concentration, two biopolymers can co-exist in a single phase whereas at

play an important role towards the solubility of "protein-polysaccharide" aggregates.

higher concentration, it starts phase separation. (Fig. 3).

BSA pH > P*<sup>I</sup>* Heating

pH < P*I*

Particle Binding


**Figure 2.** Interaction between polysaccharide and protein at various pH.




Heating Cooling


Anionic polysaccharide




Unfolded BSA

CH2 NH3 O C=O

+


Chain Segment Binding

**Figure 3.** Schematic representation of the possible mode of interaction between polysaccharides and proteins.
