Impacts of preparation conditions on the structure and emulsifying properties of casein-alginate conjugates produced by transacylation reaction

Highlights

• NaCas-alginate conjugates were prepared using the transacylation reaction.

• Increasing NaCas:PGA mass ratio resulted in a higher NaCas content of the conjugate.

• The conjugates resulted in stable emulsions with a low surfactant-to-oil ratio.

• The NaCas content had the predominant role in determining the droplet size.

• The alginate moiety stabilized emulsions with electrostatic and steric mechanisms.

Abstract

The Maillard reaction is often used to glycate proteins but produces undesirable byproducts. In this study, the transacylation reaction was used for the first time to prepare protein-polysaccharide conjugates from sodium caseinate (NaCas) and propylene glycol alginate (PGA) as novel emulsifiers. By mixing NaCas and PGA (1% w/v) at mass ratios of 1:2, 1:1, and 2:1 for 2 h with pH maintained at 11.0, NaCas-alginate conjugates with 52.8%, 66.2%, and 76.5% NaCas were prepared, respectively. The purified conjugates resulted in the preparation of oil-in-water emulsions with a low surfactant-to-oil ratio of 0.75:100 (w:v), and the resultant emulsions were stable against environmental stresses of pH, ionic strength, and thermal pasteurization. Structural analyses showed the role of NaCas content in reducing droplet size and the role of the alginate moiety stabilizing oil droplets via the electrostatic and steric mechanisms. This work may be significant to prepare protein-polysaccharide conjugates with high emulsifying capacity and tunable functionalities using a scalable and green method.

Introduction

Emulsions are important to numerous food, pharmaceutical, cosmetic and consumer products. Compared with small molecular surfactants such as Tweens and Spans, polymeric emulsifiers can form viscoelastic films on the surface of oil droplets important to the stability of emulsion droplets against flocculation and coalescence [1]. Although polymers can be synthetized to have excellent emulsifying properties, the drive for sustainability, environmental friendliness, biodegradability, and safety turns attentions to natural biopolymeric emulsifiers. Many proteins are amphiphilic and function as good emulsifiers, but the stability of droplets prepared with proteins is highly sensitive to environmental factors [2]. Near to the isoelectric point (pI) of protein or at high ionic strength, protein-stabilized emulsions are not stable because the reduced surface net charge can cause the aggregation of protein-coated oil droplets. Conversely, glycated proteins integrate the emulsifying properties of the protein moiety to form thick interfacial films and the carbohydrate moiety to stabilize oil droplets against aggregation, particularly for ionic polysaccharides that can provide strong steric and electrostatic repulsions [2].

Some polysaccharides are conjugated with polypeptides naturally or during separation and thus have emulsifying properties, such as gum arabic, sugar beet pectin, and soluble soybean polysaccharide (SSPS) [3]. However, a relatively high surfactant-to-oil ratio (SOR) is required for these polysaccharides to prepare emulsions [4], [5], due to their limited polypeptide content which is usually no more than 10% for commercial products [6], [7]. In addition, gum arabic is primarily produced in Sudan and geopolitical uncertainty has raised supply concerns [8]. Protein-polysaccharide conjugates with a relatively high content of protein can be synthesized with the Maillard reaction, and these conjugates have been proposed to substitute polysaccharide emulsifiers such as gum Arabic [9]. However, conjugation with the Maillard reaction usually needs heating treatment for several hours or even a few days at controlled temperature, time, pH, and moisture [9], [10], [11], and the complex series of reactions continue during storage and also produce numerous byproducts that cause negative sensory properties and possibly toxicity [12], [13].

Propylene glycol alginate (PGA) is an anionic polysaccharide produced from the esterification reaction of alginic acid and propylene oxide, with the esterification degree dependent on reaction conditions [14]. Under alkaline conditions, the ester bonds of PGA are cleaved to produce alginate and propylene glycol; if proteins or other polysaccharides are present, transacylation reactions occur and result in formation of covalent bonds between de-esterified carboxyl groups of PGA and amino or hydroxyl groups of proteins [15], [16]. The protein-alginate conjugates produced in the transacylation reaction have a similar structure to the Maillard conjugates. In addition, the transacylation reaction is fast, requires only the base and acid used to control pH, and does not produce byproducts, which are advantageous when compared with the Maillard reaction.

The transacylation reaction between proteins and PGA was firstly reported in 1970 and has been utilized to attach alginate onto protein-based particles or emulsion droplets [15], [17], [18]. Relevant to the present study, emulsion gels were studied using mixtures of sodium caseinate (NaCas) and alginate by acidification to induce aggregation of NaCas or addition of divalent calcium ions to bridge alginate [19], [20]. In our earlier study, ternary physical nanocomplexes prepared with NaCas, PGA, and zein were capable of preparing gel-like high internal phase emulsions with an oil fraction of 80%, but the emulsions with lower oil contents were unstable [20]. None of these studies indicate the possibility of preparing stable fluid emulsions using the physical mixtures of NaCas and alginate. In our recent study [21], the transacylation reaction was used to react PGA with individual α‐, β‐, and κ‐caseins at pH 11.0 and a casein:PGA mass ratio of 1:2, and the conjugates after purification with dialysis had a protein content of 39.17%, 37.78%, and 23.14%, respectively. The protein content of conjugates, decided by the casein type, not the casein structure was critical to the droplet size of emulsions. Emulsions prepared with these conjugates had a low SOR of 1:100 (w:v) and were stable during storage and at wide ranges of pH and ionic strength. Casein-alginate conjugates are therefore promising novel biopolymeric emulsifiers.

In the present study, we hypothesize the protein content of casein-alginate conjugates can be improved by adopting NaCas and controlling the NaCas:PGA mass ratio during the transacylation reaction. Since NaCas is a commercial ingredient with all types of caseins, the present study may improve not only interfacial properties but also practical applications of the conjugates. The first specific objective was to study the effect of NaCas:PGA mass ratio during the transacylation reaction on the molecular structure of casein-alginate conjugates. The formation of covalent bonds was confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SEC-HPLC). After purification with dialysis and lyophilization, the conjugates were characterized with Fourier-transform infrared (FTIR) spectroscopy, protein content, surface hydrophobicity, atomic force microscopy (AFM), zeta (ζ)-potential, and monolayer thickness after adsorption on polystyrene latex beads. The second objective was to study emulsifying properties of the casein-alginate conjugates with different protein and polysaccharide contents. Oil-in-water (O/W) emulsions were prepared from the conjugates, with comparison to individual constituent biopolymers and their mixtures. Emulsions were characterized for visual appearance, microstructures using optical microscopy and confocal laser scanning microscopy (CLSM), and the stability against different environmental factors and during 28-d storage.