**1. Introduction**

The emulsions stabilized by aqueous solid particles are termed Pickering emulsions, which is different from those stabilized by small-molecule surface emulsifiers. Solutions of many organic ingredients, such as protein, glue, flour, milk, starch, saponin, could act as emulsifiers for Pickering emulsion [1]. Pickering emulsions prepared using food-grade colloidal particles can be designed to have high physical stability and overcome the problems of low biocompatibility [2] and environmental pollution [3] caused by traditional synthetic surfactants. With the increasing awareness and pursuit of health and safety from consumers, formulating food with natural materials has been increasingly expected from academia to the food industry. Protein-based colloidal particles are particularly suitable since they are widely available, inexpensive, and show great nutritional benefits [4]. Re-

**Citation:** Li, Y.; Xu, G.; Li, W.; Lv, L.; Zhang, Q. The Role of Ultrasound in the Preparation of Zein Nanoparticles/ Flaxseed Gum Complexes for the Stabilization of Pickering Emulsion. *Foods* **2021**, *10*, 1990. https:// doi.org/10.3390/foods10091990

Academic Editor: Jayani Chandrapala

Received: 17 July 2021 Accepted: 21 August 2021 Published: 25 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cently, there are many pieces of research on Pickering emulsions using different colloidal particles, such as starch [5], polysaccharides [6], protein [7], and phospholipids, to form combinations to pursue the emulsification effect closer to that of synthetic chemical surfactants [8]. However, research focusing on the influence of the emulsion preparation process on emulsion stability is still far from enough.

Many techniques have been applied for emulsion preparation, such as high shear mixers (homogenization), ultrasound [9] micro-fluidization, and high-pressure homogenization [10]. Homogenization and ultrasound are the two most commonly used methods to prepare Pickering emulsions. Ultrasound has the advantages of high efficiency, good reproducibility, low cost, simple operation, no pollution, and being able to maintain the internal nutrients of food, which makes the ultrasonic technology widely used. Many reports show ultrasonic treatment could improve the emulsification property of the emulsion stabilized by soy protein isolate [11], chitosan [12], and whey protein-pectin complexes [13]. Moreover, high-intensity ultrasound is more energy-efficient than high-pressure homogenization and microfluidizer [14]. However, the effects of ultrasound on the stability of emulsion were rarely characterized in detail, e.g., effects of ultrasonic parameters, substrates, different processing procedures. In addition, there are few studies on the interaction between different emulsion preparation methods.

In our previous study, we found ultrasonic pretreatment could improve the stability of zein stabilized Pickering emulsion. But if ultrasound is directly applied to zein nanoparticles (ZNP), which is prepared by anti-solvent precipitation method, the stability of ZNP would not have been improved after ultrasonic treatment [15]. Julius W. J. de Folter et al. demonstrated that ZNP system synthesized using anti-solvent precipitation procedure is very stable, specifically at a pH of 3–4. However, Pickering emulsions stabilized by ZNP failed to form stable emulsions due to extreme environmental stresses (pH, heating, and salt) [16]. To improve the stability of the ZNP emulsion, forming a complex with other hydrophilic molecules, such as hydrophilic protein, polysaccharides, or polyphenols, is a common method. Among them, polysaccharides, including sodium alginate, low-acyl gellan gum, gum arabic, chitosan, and carboxymethyl dextrin have been reported to be effective [17–20]. Flaxseed gum (FSG), with good gelling, emulsifying, and rheological properties, was used to form complexes with ZNP [21–24]. Although our previous research showed that flaxseed gum could improve the stability of ZNP emulsion, the influences of different preparation methods and different combinations of ultrasound and homogenization on emulsion have not been studied. Therefore, in the current study, we studied the preparation of zein nanoparticles-flaxseed gum (ZNP-FSG) complexes according to different procedures, including either applying ultrasonication and/or homogenization of the mixed solution without any previous treatment or pretreatment using ultrasonic of one or both ingredients. Both the ZNP-FSG complexes and the final emulsions were characterized. The aim is to devise the preparation route of ZNP-FSG complexes that are best suited for the stabilization of Pickering emulsions [25].

#### **2. Materials and Methods**

#### *2.1. Materials*

Zein was purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Flaxseed gum was provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China). Corn oil was obtained from a local supermarket (Nanjing, China). Other reagents were analytical grade.

#### *2.2. Preparation and Pretreatment of Samples*

In order to analyze the role of ultrasound on Pickering emulsion preparation, different combinations of ultrasound and homogenization and different ultrasound ingredients were compared, 1. zein, 2. FSG, 3. zein-FSG mixtures.
