Elsevier

Chemical Physics

Volume 538, 1 October 2020, 110910
Chemical Physics

Electrostatic-driven structural transformation in the complexation of lysozyme and κ-carrageenan

https://doi.org/10.1016/j.chemphys.2020.110910Get rights and content

Highlights

  • Evolution route of the LYS/κ-CRG complexes during acidification is revealed.

  • Role of the electrostatic interactions is clearly elucidated.

  • Viscometric property of the LYS/κ-CRG aqueous mixture is investigated.

  • Thermal stability of LYS is improved by complex coacervates formation.

Abstract

Lysozyme (LYS) and κ-carrageenan (κ-CRG) are important components widely used in food and pharmaceutical products. In this work, we investigated the interactions in the aqueous mixtures of LYS and κ-CRG and the structural transformation of the LYS/κ-CRG complexes. When co-soluble polymers and soluble complexes are formed in near-neutral environment, insoluble complex coacervates are formed at lower pHs. Significant accumulation and size expansion of coacervate particles occur at pH = 4.0 where maximum interactions are presented at the mixing ratio of LYS/κ-CRG = 2:1. The coacervate system is flexible to rearrange to reach a charge balanced state by gradual protonation of LYS and κ-CRG. Addition of NaCl up to 50 mM weakens the electrostatic attractions among the aggregates through counterion binding. Thermal stability of LYS by forming complex coacervates differs from that in water, and effective protection of protein is achieved as the unfolding temperature is increased by 9 °C.

Graphical abstract

Complexation of lysozyme and κ-carrageenan can be controlled by tuning the acidity.

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Introduction

Complex coacervation is an important approach to fabricate functionalized and complicated structures in food, pharmaceutical and cosmetic industries [1]. Microcapsules composed of biopolymers through complex coacervation have been extensively applied to contain and deliver targeted bioactive compounds in the processes of structure formation and stabilization, drug delivery and controlled release, due to their outstanding biosafety, biocompatibility and bioactivity [2], [3], [4], [5], [6], [7]. By tuning conditions such as concentration, acidity and ionic strength, different protein/polysaccharide complexes possessing specific morphology, quantity and property can be obtained.

In aqueous solutions, the mixing of polysaccharide with protein may produce a few kinds of specific structures [8], producing a thermodynamically incompatible system. Since protein and polysaccharide are usually electrically charged, the competition between attractive and repulsive electrostatic interactions among polymers plays a critical role in constructing different structures, such as aqueous two-phase systems and insoluble complex coacervates [9], [10], [11], [12], [13]. Generally, soluble complexes are formed when the electrostatic attractions are not significant, while too strong attractions usually cause the precipitation of both biopolymers by forming insoluble complex coacervates. Souza et al. [1] investigated the interactions between ovalbumin and carrageenan (which is a mixture of κ and λ types) in aqueous solution with the presence of 0.01 M NaCl, and different morphologies of complexes were observed with the changing acidity. Antonov et al. [14] reported an investigation of the effects of ratio of κ-CRG to LYS and ionic strength on complexation of lysozyme (LYS) and κ-carrageenan (κ-CRG) governed by electrostatic interactions and secondary forces. Reports by Liu et al. [15], Dowling et al. [16] and Seyrek et al. [17] also discussed the influences of acidity and ionic strength on the formation of protein/polysaccharide complexes. These specifically fabricated structures are facilely obtained starting from inexpensive materials, and some of them have already been applied in industrial practices for the purpose of, for example, improving food texture and drug stability [18]. Although progresses made by researchers have explained the mechanism of complex formation to a certain extent, and both experimental and computational methods have been applied [19], [20], [21], [22], the database still needs enrichment and the structural transitions of complexes need to be elucidated for development and application of the desired functional structures.

Recently we fabricated the ovalbumin/κ-carrageenan (OVA/κ-CRG) and LYS/κ-CRG colloid particles to act as carriers for curcumin delivery, and the interactions between protein and polysaccharide have been investigated [23], [24], [25]. The controlled release of curcumin was achieved, and the thermal and light stabilities and antioxidant activity of curcumin were impressively improved by encapsulation. In this work, we attempt to provide insights into the mechanism driving the complexation of LYS with κ-CRG in water under the effects of acidity and composition. The structural transition of the LYS/κ-CRG complexes at the mass ratio of LYS to κ-CRG of 2:1 has been revealed. The changes in thermal stabilities by forming complex coacervates are also discussed.

Section snippets

Materials

LYS (from egg white), κ-CRG (drying loss 0.080 in mass fraction, ash content 0.220), Na2HPO4 (0.980), NaCl (0.998) and citric acid (0.980) were purchased from Aladdin Co., ltd. (China). HCl (0.365) was purchased from Sinopharm (China) and diluted to 0.5 M and 6.0 M with water. The reagents were stored in the environments suggested by the suppliers and are used as received. The Direct-Q 5 UV instrument (Millipore, Germany) was used to prepared ultrapure water.

κ-CRG can be regarded as the

Formation and characterization of LYS/κ-CRG complexes

The polysaccharide, κ-CRG, is a typical kind of anionic biopolymer and usually carries negative surface charge. The protein, LYS, has an isoelectric point (pI) of 10.7 and it carries positive charges at a lower pH. Therefore, tuning the acidity of the solution can be an effective way to acquire suitable interactions between LYS and κ-CRG. Usually, co-soluble polymers and homogeneous gel can be obtained when the interactions are weak, and insoluble precipitates of both polymers are formed when

Conclusions

The understanding of interactions and structures of complexes formed by protein and polysaccharide is of great importance in the applications. The current study has investigated the effects of several factors including pH, mixing ratio of LYS and κ-CRG, and addition of inorganic salt on complexation. By acidification, transition points which correspond to various structure-forming events are determined and the structural transformation of the complexes is revealed. Soluble complexes are formed

CRediT authorship contribution statement

Xiaoxing Lu: Writing - review & editing, Funding acquisition, Supervision. Shaoxia Xie: Writing - original draft, Validation. Lihong Wang: Conceptualization, Investigation. Hujun Xie: Resources, Funding acquisition, Supervision. Qunfang Lei: Funding acquisition, Project administration. Wenjun Fang: Resources.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 21703205 and 21773209), the Natural Science Foundation of Zhejiang Province (No. LY17B050001), and Natural Science Foundation of Guangdong Province (No. 2020A1515010546).

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