Effects of dual succinylation and ultrasonication modification on the structural and functional properties of ovalbumin

https://doi.org/10.1016/j.foodres.2023.112511Get rights and content

Highlights

  • Succinylated OVA (S-OVA) and succinylated-ultrasonicated OVA (SU-OVA) were prepared.

  • Succinylation increased OVA structural flexibility and decreased OVA aggregation.

  • Ultrasonication enhanced the interfacial and emulsifying properties of S-OVA.

  • SU-OVA produced emulsions with lower particle size and superior stability.

  • SU-OVA expanded the application range of OVA in the protein emulsion industry.

Abstract

In this study, the functional properties of ovalbumin (OVA) were improved through dual modification with succinylation (succinylation degrees of 32.1 % [S1], 74.2 % [S2], and 95.2 % [S3]) and ultrasonication (ultrasonication durations of 5 min [U1], 15 min [U2], and 25 min [U3]), and the changes in protein structures were explored. Results showed that as the succinylation degree was increased, the particle size and surface hydrophobicity of S-OVA decreased by the maximum values of 2.2 and 2.4 times, respectively, causing emulsibility and emulsifying stability to increase by 2.7 and 7.3 times, respectively. After ultrasonic treatment, the particle size of succinylated–ultrasonicated OVA (SU-OVA) had decreased by 3.0–5.1 times relative to that of S-OVA. Moreover, the net negative charge of S3U3-OVA had increased to the maximum value of − 35.6 mV. These changes contributed to the further enhancement in functional indicators. The unfolding of the protein structure and the conformational flexibility of SU-OVA were illustrated and compared with those of S-OVA via protein electrophoresis, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, and scanning electron microscopy. The dually modified OVA emulsion (S3U3-E) presented small droplets (243.33 nm), reduced viscosity, and weakened gelation behavior that were indicative of even distribution, which was visually proven by confocal laser scanning microscopy images. Furthermore, S3U3-E exhibited favorable stability, a particle size that was almost unchanged, and a low polydispersity index (<0.1) over 21 days of storage at 4 °C. The above results demonstrated that succinylation combined with ultrasonic treatment could be an effective dual modification method for enhancing the functional performance of OVA.

Introduction

Ovalbumin (OVA), which has the molecular weight of 45 kDa and comprises 386 amino acids, accounts for the highest proportion (54 %) of egg white protein (Abeyrathne, Lee, & Ahn, 2013). The amphiphilic structure (possessing hydrophobic and hydrophilic groups) of OVA has considerable effects on the functional properties (e.g., solubility, emulsibility, oil absorption capacity, and gelation) that contribute to the adjustment of the sensory and textural properties and stability of food systems (Xiong, Li, Li, & Wang, 2019). The improvement in emulsification is mostly reflected by the decline in the tension of the oil–water interface and the formation of an adsorption layer. These effects help limit the dispersion and aggregation of oil droplets (Yang et al., 2018). Emulsions can be produced via the mechanical shearing of oil and water phases and provide added functionality. However, this method forms thermodynamically unstable dispersed systems. Protein emulsions provide stability driven by dynamic interfacial adsorption (Lian et al., 2023). Native OVA has poor emulsibility and limited applications in food protein emulsion systems because its internal structure contains numerous hydrophobic residues (Wang et al., 2019).

Over the past few years, many studies have attempted to improve the emulsification properties of OVA via different protein modification techniques, including physical methods (Xiong, Li, Li, & Wang, 2019), chemical methods (Xiong, Zhang, & Ma, 2016), and biological methods (Zheng, Chang, Luo, Teng, & Chen, 2022; Campbell, Raikos, & Euston, 2003). Physical modification involves the use of a physical force field that can transform protein structures. This method usually results in the size reduction, unfolding, or denaturation of proteins (Akharume, Aluko, & Adedeji, 2021). Although it would provide highly efficient emulsifying performance, high-pressure-assisted modification is difficult to popularize due to its high installation and maintenance costs. Chemical methods are based on reactions with chemical substances. They result in either bond breakage or formation, thus subsequently changing the structure of the native protein. Biological modifications involving enzymolysis and fermentation improve protein functions under mild conditions. Although enzymatic hydrolysis is an environmentally friendly modification method, its excessively long reaction time increases the risk for microbial growth.

Among protein modification methods, succinylation (Hu et al., 2022) and ultrasonication (Xiong et al., 2016) are respectively regarded as simple and efficient physical and chemical modification methods and have been extensively used to enhance the functional properties of OVA. Succinylation is one of the most common chemical methods for the modification of food proteins; in this process, positively charged lysine groups in proteins are replaced with negatively charged succinyl groups (Basak & Singhal, 2022). In OVA, hydrophobic tails are directed toward the oil medium, whereas negatively charged hydrophilic groups are directed toward the water phase. The increase in the electronegativity of the hydration layer around oil droplets with the increment in the protein concentration in the aqueous phase prevents the flocculation and coalescence of the oil droplets (Basak & Singhal, 2022). Accordingly, the increment in electronegativity is beneficial to the unfolding of the protein structure and the improvement in emulsification properties. For example, the emulsifying properties of chicken liver protein subjected to pH-dependent succinylation remarkably improved (Lu et al., 2021). Succinylated Antarctic krill proteins demonstrated a similar phenomenon (Wang et al., 2022). A previous work reported on the gel properties of succinylated OVA (S-OVA) but failed to mention the effects of succinylation on emulsification properties (Hu et al., 2022).

Ultrasonication, a typical nonthermal physical processing technique with a special cavitation and mechanical effect, has been extensively used to modify food proteins (Higuera-Barraza, Toro-Sanchez, Ruiz-Cruz, & Marquez-Rios, 2016). The cavitation effect is a cyclical process caused by ultrasound wherein bubbles expand rapidly and then disintegrate violently. Powerful mechanical forces, such as shear stress and turbulence, can develop during this process (Zhang et al., 2022). As a result, the turbulent flow of protein molecules, along with the fracture of covalent bonds in polymeric chains, contributes to protein adsorption at the oil–water interface and accordingly enhances emulsification properties (Kamani, Semwal, & Khaneghah, 2022). Some previous studies reported that ultrasonication could significantly increase the emulsibility and emulsifying stability of myofibrillar proteins (Chen, Zhang, Xue, & Xu, 2020), soybean proteins (Li et al., 2020), and dairy proteins (O’Sullivan, Arellano, Pichot, & Norton, 2014). Furthermore, although Xiong (Xiong, Li, Li, & Wang, 2019; Xiong et al., 2016) had reported on the structure, interface, and gelation properties of ultrasonicated OVA, the emulsification-related properties of this protein have not been investigated in detail.

Although a large number of studies on protein modification technology have been reported in the past few years, single modification methods cannot guarantee excellent functional properties because they still have some certain technical defects. Hence, researchers have been inspired to develop different dual modification approaches based on single methods to further improve the hydration, surface activity, and rheological properties of proteins (Kamani et al., 2022). Dual modification is defined as a combination of two single methods in different orders and repetitions (double physical/chemical/biological, physical–chemical, and physical–biological methods) (Kamani et al., 2022). Succinylation has been combined with enzymolysis (Pan et al., 2019) and glycosylation (Liu et al., 2022) to improve emulsifying properties. Ultrasonication has been extensively combined with other modification processes, such as heating (Zhong & Xiong, 2020), acid induction (Huang, Ding, Li, & Ma, 2019), and transglutaminase treatment (Ahmadi, Razavi, & Varidi, 2017). However, no research has been conducted on the dual modification of OVA through succinylation and ultrasonication to improve its emulsification properties.

In this study, the effects of different degrees of succinylation combined with different durations of ultrasonication on the structural, physicochemical, and functional properties of OVA were evaluated. Furthermore, succinylated–ultrasonicated OVA (SU-OVA) was applied in the preparation of emulsions, and its emulsion performance was examined on the basis of its particle size, rheological behavior, microstructure, interfacial protein adsorption, and storage stability to expand the practical application of OVA in the food industry. A schematic of the preparation of SU-OVA is shown in Scheme 1.

Section snippets

Materials

OVA (62 %–88 %) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Succinic anhydride (SA, AR, 98 %) was purchased from Macklin Biotechnology Co. ltd. (Shanghai, CN). Sunflower seed oil was procured from COFCO Group Co. ltd. (Harbin, Heilongjiang, CN). All other chemicals and reagents used were of analytical grade. The BCA protein assay kit used in this work was purchased from Beyotime Biotechnology (Shanghai, CN).

Treatment of OVA through succinylation and ultrasonication

S-OVA was prepared by following a previously reported method (Pan et al.,

DS

DS was calculated by measuring the absorbance of a compound on the basis of the reaction between ninhydrin and the amino acids of a protein. The DS values of S-OVA with different SA:OVA mass ratios are displayed in Fig. 1. DS increased sharply to 89.5 % as the SA:OVA mass ratio was increased from 1 % to 7 %. Then, despite the continuous increment in SA, the increment in DS gradually decelerated until no significant difference was observed at the SA:OVA mass ratio of 9 %. The same phenomenon was

Conclusion

This study illustrated that dual modification at different DS (32.1 %, 74.2 %, and 95.2 %) combined with different ultrasonication durations (5, 15, and 25 min) considerably improved the physicochemical and functional properties of OVA. Succinylation reduced particle sizes and H0 and increased net negative charge and SH group contents. Hence, the EAI and ESI of S-OVA were 2.7- and 7.3-fold higher than those of OVA, respectively. The subsequent ultrasonic treatment enhanced emulsibility and

CRediT authorship contribution statement

Ru-yi Zhang: Investigation, Formal analysis, Visualization, Writing – original draft, Data curation. Yang Wang: Resources, Visualization. Yi Jiang: Data curation, Software. Er-hu Min: Validation, Formal analysis. Sheng-qi Rao: Funding acquisition, Project administration, Conceptualization, Methodology, Writing – review & editing.

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 research was supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJA550002), the China Postdoctoral Science Foundation (2018M632393), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_3231), and the Foundation of China National Key Research & Development Program (2018YFD0400303).

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