**1. Introduction**

Emulsions are widely used in many industries and have an important role in the food industry to stabilize many different formulations that have either oil-in-water (O/W) or water-in-oil (W/O) dispersions [1]. Stability of emulsions is influenced by many factors,

**Citation:** Smułek, W.; Siejak, P.; Fathordoobady, F.; Masewicz, Ł.; Guo, Y.; Jarz˛ebska, M.; Kitts, D.D.; Kowalczewski, P.Ł.; Baranowska, H.M.; Stangierski, J.; et al. Whey Proteins as a Potential Co-Surfactant with *Aesculus hippocastanum* L. as a Stabilizer in Nanoemulsions Derived from Hempseed Oil. *Molecules* **2021**, *26*, 5856. https://doi.org/10.3390/ molecules26195856

Academic Editor: Seyed Fakhreddin Hosseini

Received: 2 July 2021 Accepted: 20 September 2021 Published: 27 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

e.g., the concentration and ratio of individual phases, methods of preparation, storage condition, as well as the presence of compounds demonstrating the surface-active property (stabilizers) [2]. Therefore, new sources of natural, effective stabilizers, or surfactants, are in high demand to use plant-based ingredients that provide bioactive extracts [3–5], hydrocolloid activity [6], plant proteins [7–9]. Many biopolymers (proteins, polysaccharides or the combinations) that are used to assist in the formation of an emulsion have excellent effectiveness since they contain both hydrophilic and hydrophobic groups capable of lowering the surface tension between water and the oil phase [10]. Such proteins as collagen, whey protein, soy protein isolate and casein are the most commonly used due to a high emulsifying index and the water/oil holding capacity [11].

Hempseed oil (HSO), derived from the seeds of *Cannabis sativa* L., is recognized for both nutritional, health-promoting and bioactive properties [12,13]. It is a good source of both n-3 and n-6 essential fatty acids, specifically linoleic acid (55%) and alpha-linolenic acid (20%), with concentrations relatively higher than in other vegetable oils [14]. HSO is also a source of gamma linoleic acid (GLA) not found in many vegetable oils, but with noted bioactive properties. HSO's health advantages have been ascribed to the 3:1 n-3/n-6 fatty acid ratio, which is an established optimum for human nutrition uptake [14], and the presence of GLA [15]. Additionally, the presence of minor components, such as tocopherol, tocotrienols, carotenes, minerals, terpenoids and β-sitosterol also add to the nutritional value of HSO. Besides these health effects, HSO is often used in formulations that require high miscibility with water [16], which reduces the reliance of additional surfactants for use in nanoemulsions. Accordingly, HSO has been used in cosmetic, nutraceutical, food and functional food industries.

Dairy proteins have been widely used as emulsifiers in food industry. They adsorb to the oil droplet interface, producing a consistent protective film that can help prevent droplet aggregation [17]. They have also been used as emulsifiers in nanoemulsion formulations. Whey protein (WP), with a noted high nutritional value, is often used as an ingredient in many food products and for the production of sports nutrition [18,19]. Whey proteins are a good source of essential amino acids, and globular proteins isolated from whey are noted for having biological activities: antioxidant, immunostimulatory and anti-obesity [20]. They are also effective emulsifiers in food formulations because their amphiphilic properties enable relatively stable gels and microcapsules without additional chemical additives. For example, WP has been used to encapsulate probiotics [21–23]. Iqbal et al. [24] used the WI in the formation to generate a three-dimensional network in an oil/water phase. However, the resulting emulsions did not exhibit ideal plastic behavior. Since WP can be prepared as an isolate or a concentrate, the WPI has been used to stabilize nanoemulsion formulations containing peanut oil, corn oil, β-carotene in sunflower oil and α-Tocopherol in palm oil. It was found that the lowest degradation of β-carotene at 55 ◦C occurred when WPI was used as stabilizer in the nanoemulsion system. This was probably related to the substantial antioxidant property of WPI [25].

*Aesculus hippocastanum* L. is also known as horse chestnut [26] and contains many biologically active compounds including escin, a mixture of saponins with medicinal properties. The health beneficial effects of AH include anti-inflammatory and anti-edematous [27] properties, with potential benefits for kidney disease in diabetic nephropathy [28]. Due to the high content of polyphenols, such as quercetin and kaempferol [29], *Aesculus hippocastanum* L. bark also exhibits antioxidant and antitumor activities [30]. In addition, the extracts from *Aesculus hippocastanum* L. (AHE) with a high content of saponins [31] exhibited high interfacial activity due to hydrophobic aglycone and hydrophilic sugar residues [12]. The United States Department of Agriculture (USDA) considers the AHE to be generally regarded as safe, and the European Union has also approved it for use as a foam stabilizer in beverages [12]. AHE was found to be an effective stabilizer when used as a stabilizer in hempseed oil (HSO) nanoemulsions [12]. HSO–nanoemulsion formulations containing 2 g/L AHE also produce smaller droplets.

The purpose of this study was to prepare an O/W formulation of an HSO nanoemulsion system that was stabilized with an optimal concentration of the WPI and the AHE. The goal was to obtain both acceptable stability of the emulsion and also potential biological activity of its ingredients for both food and biomedical applications. We also focused on using the WPI as a potential cosurfactant.
