*Article* **Whey Proteins as a Potential Co-Surfactant with** *Aesculus hippocastanum* **L. as a Stabilizer in Nanoemulsions Derived from Hempseed Oil**

**Wojciech Smułek <sup>1</sup> , Przemysław Siejak <sup>2</sup> , Farahnaz Fathordoobady <sup>3</sup> , Łukasz Masewicz <sup>2</sup> , Yigong Guo <sup>3</sup> , Małgorzata Jarz˛ebska <sup>4</sup> , David D. Kitts <sup>3</sup> , Przemysław Łukasz Kowalczewski <sup>5</sup> , Hanna Maria Baranowska <sup>2</sup> , Jerzy Stangierski <sup>6</sup> , Anna Szwajca <sup>7</sup> , Anubhav Pratap-Singh 3,\* and Maciej Jarz˛ebski 2,\***


**Abstract:** The use of natural surfactants including plant extracts, plant hydrocolloids and proteins in nanoemulsion systems has received commercial interest due to demonstrated safety of use and potential health benefits of plant products. In this study, a whey protein isolate (WPI) from a byproduct of cheese production was used to stabilize a nanoemulsion formulation that contained hempseed oil and the *Aesculus hippocastanum* L. extract (AHE). A Box–Behnken experimental design was used to set the formulation criteria and the optimal nanoemulsion conditions, used subsequently in follow-up experiments that measured specifically emulsion droplet size distribution, stability tests and visual quality. Regression analysis showed that the concentration of HSO and the interaction between HSO and the WPI were the most significant factors affecting the emulsion polydispersity index and droplet size (nm) (*p* < 0.05). Rheological tests, Fourier transform infrared spectroscopy (FTIR) analysis and *L\*a\*b\** color parameters were also taken to characterize the physicochemical properties of the emulsions. Emulsion systems with a higher concentration of the AHE had a potential metabolic activity up to 84% in a microbiological assay. It can be concluded from our results that the nanoemulsion system described herein is a safe and stable formulation with potential biological activity and health benefits that complement its use in the food industry.

**Keywords:** nanoemulsion; whey protein; *Aesculus hippocastanum* L.; hempseed oil; emulsion stability; droplet size
