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Shear rheological properties of acid hydrolyzed insoluble proteins from Chlorella protothecoides at the oil-water interface

https://doi.org/10.1016/j.jcis.2019.05.029Get rights and content

Abstract

Microalgae are promising protein sources due to their overall high protein content. The low aqueous-solubility of microalgae proteins, however, limits their application in food, pharmaceutical or personal care systems, unless solubility is enhanced by e.g. hydrolysis. In this study, we examined the interfacial rheological properties at the oil-water interface of insoluble microalgae protein-rich fraction from Chlorella protothecoides and their hydrolysates prepared by hydrolysis in hydrochloric acid at 65 °C (Hydrolysates 65) and 85 °C (Hydrolysates 85). Results showed increased interfacial activity of the insoluble microalgae protein-rich fraction after hydrolysis: Hydrolysates 65 and Hydrolysates 85 had higher interfacial storage Gi′ and loss moduli Gi″ compared to the untreated insoluble microalgae protein-rich fraction. Increasing amounts of soluble protein fragments mixed with insoluble protein particles in hydrolysates stabilized interfacial layers. The influence of pH on the interfacial behavior of samples was also determined and revealed that Gi′ and Gi″ values of treated and untreated protein fractions decreased as pH increased beyond their isoelectric points due to increasing electrostatic repulsions between adsorbed protein fragments and aggregates. The high viscoelasticity of the acid-hydrolyzed insoluble microalgae protein-rich fraction at the oil-water interface indicates a high potential for them to be useful in stabilizing emulsion-based products.

Introduction

Plant proteins are regarded as sustainable protein sources compared to animal proteins due to their lower resource consumption [1]. Microalgae similarly promise to serve as novel sustainable sources for proteins due to the ability to rapidly grow in closed reactor systems where water and unused nutrients can be recirculated and environmental impacts can be minimized. More importantly, though, many microalgae species express high amounts of proteins requiring in principal less effort to obtain protein concentrates for subsequent applications. For example, studies reported as much as 68% (dry weight) of protein in Chlorella species, and these proteins were found to be of a high nutritional content [2], [3]. However, more than half of the proteins in Chlorella are water-insoluble making their application in food, pharmaceutical or personal care products difficult. Their lack of technofunctionality prevents them from being readily used to stabilize emulsions or foams or to manufacture gels. In a previous study, thermal-acid hydrolysis was used to increase the solubility of an insoluble microalgae protein-rich fraction obtained from Chlorella protothecoides. The solubility of the insoluble microalgae protein-rich fraction increased after hydrolyzing at 65 °C (Hydrolysates 65) and 85 °C (Hydrolysates 85) for 4 h in 0.5 M hydrochloric acid (HCl) (Table 1). This increase in solubility was attributed to an acid-induced fragmentation of protein-rich particles into smaller aggregates and medium- to short-chain peptides. The decomposition of insoluble protein particles to soluble protein fragments makes it theoretically possible to use the treated fractions as functional ingredients in the formulation of a variety of products such as foods, beverages, creams, etc. To that purpose, it is of interest to better understand their ability to stabilize oil-water or gas-water interfaces in order to manufacture food dispersions such as oil-in-water emulsions or foams. Aside from adsorption properties (e.g. adsorption kinetics, or equilibrium interfacial coverages), interfacial rheological properties are of critical importance since food dispersions are often manufactured at high dispersed phase volume fractions where tightly packed dispersed droplets or particles require highly viscoelastic layers to be stable over time. Moreover, changes in rheological behavior over time can yield important information about the aging behavior of interfaces. In the present work, we, therefore, studied the interfacial rheological properties of an untreated insoluble microalgae protein-rich fraction and two of their hydrolysates at the oil-water (O-W) interface.

It has been reported that the interfacial rheological properties of either animal-derived or plant-derived proteins can be improved by controlled hydrolysis. Tamm and Drusch [4] reported that the interfacial adsorption of whey protein at the oil-water interface was accelerated with increasing degrees of hydrolysis. The increase in interfacial activity of the hydrolysates was attributed to the presence of lower molecular weight protein fragments having a more flexible structure, a higher surface hydrophobicity and a lower ζ-potential [5], [6]. A study of soy protein hydrolysates verified that their flexible structure and lower molecular weights resulted in large surface loads yielding interfacial films with high viscoelasticities facilitating the manufacture of stable oil-in-water emulsions [7]. Moreover, acid-deamidated wheat protein was reported to form a relative thick interfacial layer, consequently, enhancing emulsion stability against coalescence and heating [8]. However, the above-mentioned studies on interfacial activity are all about solubilized protein hydrolysates. In contrast, hydrolysates from insoluble protein-rich fractions from microalgae are heterogeneous mixtures that include both soluble protein fragments and large, partially hydrolyzed insoluble protein particles. The interfacial rheological behavior of such complex mixtures is to date though not yet known. There may be some benefits to them since both small surface-active molecules and larger aggregates providing steric-hindrances are present. In this study, we, therefore, hypothesized that the hydrolyzed microalgae protein-rich fractions (Hydrolysates 65 and Hydrolysates 85) may be able to form shear-resistant interfacial adsorption layers due to them containing increasing amounts of flexible protein fragments mixed with insoluble aggregates. To that purpose, we studied the interfacial rheological properties of the insoluble microalgae protein-rich fraction and the hydrolysates at neutral as well as at pH 3, pH 5 and pH 9.

Section snippets

Materials

Dried Chlorella protothecoides biomass was purchased from Roquette Frères (Lestrem, France). An insoluble microalgae protein-rich fraction was extracted from the biomass according to the method developed by Grossmann et al. [9]. Two hydrolysates were produced at 65 °C and 85 °C in 0.5 M HCl for 4 h according to the method published by Dai et al. [10] with modification. The physicochemical properties including protein content, solubility, isoelectric point (pI) and molecular weight distribution

Viscoelastic properties of the initial dispersion, sediment, and supernatant of untreated insoluble microalgae protein-rich fraction and the hydrolysates at pH 7

In this section, we aimed to explore the interfacial rheological properties of dispersed untreated and hydrolyzed protein-rich fractions, as well as sediments and supernatants separated from the dispersions via centrifugation at neutral pH by conducting small oscillatory time and amplitude sweeps at an oil-water interface. Time sweeps may yield insights as to the formation of interfacial networks at O-W interfaces and show when adsorption equilibria have been achieved. In subsequent interfacial

Conclusions

Proteins extracted from Chlorella protothecoides show a low water solubility, which limits their application in dispersed systems such as emulsions and foams. Therefore, studies on proteins from microalgae are mainly with the soluble protein fractions [31], [32], [33]. Acid hydrolysis of the insoluble microalgae protein-rich fraction offers a pathway to obtain soluble protein fractions, which can be used to stabilize oil-water interfaces. Our results showed that the interfacial layers become

Acknowledgments

This work was supported by the China Scholarship Council (CSC NO. 201506670001) and Bioeconomy graduate program BBW-ForWerts (200045, Baden-Württemberg, Germany). We thank support by the Swiss National Science Foundation (SNF, NO. 200021-175994) for Jotam Bergfreund.

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