Elsevier

Food Research International

Volume 72, June 2015, Pages 91-97
Food Research International

Complex coacervation between flaxseed protein isolate and flaxseed gum

https://doi.org/10.1016/j.foodres.2015.03.046Get rights and content

Highlights

  • Flaxseed protein and gum were extracted from raw flaxseed.

  • Interactions of protein and polysaccharide were studied as function of pH and different mixing ratios.

  • Different phase boundaries related to structure-forming events were determined.

  • Changes in the secondary structure of protein with lowering of pH were found facilitating the interactions of protein and polysaccharide.

  • Optimum pH and protein-to-polysaccharide ratio for complexation was found to be 3.1 and 3:1.

Abstract

Flaxseed protein isolate (FPI) and flaxseed gum (FG) were extracted, and the electrostatic complexation between these two biopolymers was studied as a function of pH and FPI-to-FG ratio using turbidimetric and electrophoretic mobility (zeta potential) tests. The zeta potential values of FPI, FG, and their mixtures at the FPI-to-FG ratios of 1:1, 3:1, 5:1, 10:1, 15:1 were measured over a pH range 8.0–1.5. The alteration of the secondary structure of FPI as a function of pH was studied using circular dichroism. The proportion of ɑ-helical structure decreased, whereas both β-sheet structure and random coil structure increased with the lowering of pH from 8.0 to 3.0. The acidic pH affected the secondary structure of FPI and the unfolding of helix conformation facilitated the complexation of FPI with FG. The optimum FPI-to-FG ratio for complex coacervation was found to be 3:1. The critical pH values associated with the formation of soluble (pHc) and insoluble (pHɸ1) complexes at the optimum FPI-to-FG ratio were found to be 6.0 and 4.5, respectively. The optimum pH (pHopt) for the optimum complex coacervation was 3.1. The instability and dissolution of FPI–FG complex coacervates started (pHɸ2) at pH 2.1. These findings contribute to the development of FPI–FG complex coacervates as delivery vehicles for unstable albeit valuable nutrients such as omega-3 fatty acids.

Introduction

The process of complex coacervation or associative phase separation in protein–polysaccharide mixtures occurs due to electrostatic attraction of oppositely charged molecules, eventually leading to a solvent-rich and a biopolymer-rich phase (Schmitt and Turgeon, 2011, Tolstoguzov, 1998). Other factors influencing the complex coacervation are charge density, relative ratio, and total concentration of biopolymers, pH, and temperature of the solvent (Schmitt, Sanchez, Desobry-Banon, & Hardy, 1998). Protein–polysaccharide mixtures form electrostatic complexes in a narrow pH range. Proteins are positively charged below their isoelectric point (pI) and can undergo complexation with negatively charged polysaccharides, resulting into soluble complex coacervates at pHc, where pHc is defined as the pH at which noncovalent interaction between protein and polysaccharide initiates (Aryee & Nickerson, 2012). Further reduction in mixture pH results into the formation of insoluble complexes at pHɸ1, where pHɸ1 is defined as the pH at which interaction between protein and polysaccharide is strong enough to cause macroscopic phase separation (Turgeon, Beaulieu, Schmitt, & Sanchez, 2003). The yield of complex coacervates is highest at pHopt, where the net charge on the system is zero. The dissolution of complex coacervates back to solution state due to the protonation of polysaccharide occurs at pHɸ2, where pHɸ2 is defined as the pH beyond which interaction between protein and polysaccharide starts decreasing. (Elmer, Karaca, Low, & Nickerson, 2011). The determination of these important pH values (pHc, pHɸ1, pHopt, and pHɸ2) for any protein–polysaccharide combinations provides better understanding of complexation behavior as a function of pH. De Kruif, Weinbreck, and de Vries (2004) suggested that pH induced changes in the conformation of protein also influence the complexation of polymers with proteins.

A number of studies have reported that plant proteins are capable of forming complex coacervates in the presence of polysaccharides. Pea protein is the most widely studied protein for complex coacervation (Ducel et al., 2004, Elmer et al., 2011, Klemmer et al., 2012, Liu et al., 2010, Liu, Low and Nickerson, 2009a, Liu, Low and Nickerson, 2009b). Other plant proteins considered appropriate for coacervation include soy protein (Jaramillo, Roberts, & Coupland, 2011), canola protein (Klassen, Elmer, & Nickerson, 2011) and corn protein (Quispe-Condori, Saldana, & Temelli, 2011).

There are plant proteins that are theoretically known to possess favorable characteristics for coacervation but are unexplored practically. Dickinson (2003) reported that charge density and droplet size are two important characteristics required for the stabilization of an emulsion. Wang, Li, Wang, Adhikari, and Shi (2010) observed that flaxseed protein concentrate when compared to soy protein concentrate, possessed higher surface charge and smaller emulsion droplet size. Recently, Kuhn, e Silva, Netto, and da Cunha (2014) found that flaxseed protein isolate (FPI) based emulsion are more stable than mixed FPI—whey protein isolate stabilized emulsions. In addition, the amino acid profile of flaxseed protein is nutritionally desirable, and it is considered nutritionally similar to other oil seed proteins such as soybean (Oomah, 2001). However, the complexation behavior of flaxseed protein with its own polysaccharide or with other polysaccharides has not been studied.

Flaxseed gum (FG) is another plant polymer identified as a good emulsifier (Cui, Ikeda, & Eskin, 2007). FG is a heteropolysaccharide composed of xylose, arabinose, glucose, galactose, galacturonic acid, rhamnose, and fucose (Cui, Mazza, Oomah, & Biliaderis, 1994). Functional properties of flaxseed gum are comparable to those of gum Arabic, and hence it can be used to replace gum Arabic in emulsions (Mazza & Biliaderis, 1989). Moreover, the consumption of flaxseed gum as dietary fiber is reported to reduce the blood glucose level thereby reducing the risk of coronary artery disease (Oomah & Mazza, 2000).

The important nutritional characteristics of flaxseed protein and gum mean that they can be economical source of functional foods (Oomah, 2001). A thorough study on the complexation behavior of these two biopolymers would help produce novel FPI–FG complex coacervates, which can be preferentially used to microencapsulate active bio-ingredients such as omega-3 oils. This study determines the optimum pH range, the FPI-to-FG ratio, and total biopolymer concentration required for the formation of soluble and insoluble complexes between FPI and FG. In order to gain greater insight into the formation of these complex coacervates, the underlying structural change of flaxseed protein as a function of pH was also investigated. Except for this work, the complexation behavior of flaxseed protein and flaxseed gum has not, so far, been reported.

Section snippets

Materials

Golden flaxseeds (Linum usitatissimum) were received from Stoney Creek Oil Product Pty. Ltd (Talbot, VIC, Australia). FG and flaxseed protein isolate (FPI) were extracted in the laboratory at Federation University, Australia. All other chemicals used in this study were purchased from Sigma-Aldrich Australia (Sydney, New South Wales, Australia) and were analytical grade.

Chemical analysis of FPI and FG

Chemical analyses of the extracted FG showed the following: 4.42 ± 0.47% moisture, 9.35 ± 0.84% protein (%N × 6.25), 1.75 ± .022% lipid, 3.17 ± 0.43% ash, and 81.31% carbohydrate.

Analysis of extracted FPI showed the following: 90.60 ± 1.31% protein, 4.2 ± 0.3% moisture, 2.20 ± 0.24% ash, and 1.06 ± 0.18% lipid and 1.94 ± 0.37% carbohydrate. All of the above results are on weight (w/w) basis.

Effect of pH on the secondary structure of flaxseed protein

Fundamental understanding of pH induced conformational changes in protein structure provides insight on complexing behavior

Conclusions

This study demonstrated that flaxseed protein isolate (FPI) and flaxseed gum (FG) can be successfully complexed at optimized conditions. The variation of pH affected the secondary structure configuration of FPI and the unfolding of helix conformation facilitated the complexation of FPI with FG at lower pH values. The optimum FPI-to-FG ratio and the pH value for complexation between FPI and FG were 3:1 and 3.1, respectively, and the resultant complex coacervates were found to be stable at low pH

Acknowledgments

The first author acknowledges the International Post Graduate Research Scholarship (IPRS) and Australian Postgraduate Award (APA) provided to her by the Australian Federal Government. The authors wish to thank Bruce Armstrong, Divya Eratte, and Bo Wang for technical help during experiments. This work was partially supported by Australian Government's Collaborative Research Network (CRN) initiative.

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