**1. Introduction**

Thymol (2-isopropyl-5-methylphenol), a monoterpene phenol, is the principal active component of essential oil obtained from thyme, which has been applied as traditional herbs in the cure of disorders affecting the digestive, cardiovascular, and nervous systems [1]. Due to the numerous functions of thymol, such as antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenesis functions, the research on its potential to be utilized as a food preservative and nutraceutical is increasing dramatically [2]. For example, Pan et al. stated that thymol could be utilized in food matrices and depolarize the microbial cytoplasmic membrane to extend the shelf life of productions [3]. Moreover, thymol is already widely applied as a natural feed additive for livestock, which could improve the metabolism and absorption of the nutrients in the animal gu<sup>t</sup> via enhancing digestive enzymes and modulating intestinal microbiota activity to increase the growth indices (including body weight, feed efficiency, and daily growth rate) of them [4–6]. Thymol has been included in the list of 'Generally Recognized As Safe' for application as food additives by the FDA, and it was registered by the European Commission for utilization in the food field due to the

**Citation:** Zhou, W.; Zhang, Y.; Li, R.; Peng, S.; Ruan, R.; Li, J.; Liu, W. Fabrication of Caseinate Stabilized Thymol Nanosuspensions via the pH-Driven Method: Enhancement in Water Solubility of Thymol. *Foods* **2021**, *10*, 1074. https://doi.org/ 10.3390/foods10051074

Academic Editors: Hong Wu and Hui Zhang

Received: 8 April 2021 Accepted: 10 May 2021 Published: 12 May 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/).

lack of intake risk [7,8]. Meanwhile, a comprehensive review has concluded and shown the safety and anticancer activity of thymol [9]. However, its commercial application is limited due to its hydrophobic nature and low water solubility [8,10]. Several types of delivery and encapsulation systems have been investigated and evaluated to enhance the water solubility of thymol; for example, Nasrabadi et al. [11] improved the water solubility and bioaccessibility of thymol via encapsulating it into Pickering emulsions; Cakir et al. [12] fabricated thymol loaded chitosan nanoparticles via ionic gelation method to overcome the limitation of thymol; and nanoliposome was applied by Heckler et al. [13] to encapsulate free thymol as antimicrobials in food products. Normally, thymol as a lipophilic element has been encapsulated in delivery systems via dissolving thymol into organic liquid firstly, which is criticized as leading to high costs and potential damage to the consumer's health. Thus, it is significant to find effective and environmentally friendly methods for handling the key application limitation and challenge of thymol encapsulation.

Recently, an easily scalable and environmentally friendly approach, termed the pHdriven method, was developed [14,15]. It has been utilized to incorporate lipophilic phenols (mainly curcumin) into a variety of delivery systems such as liposomes [16], nanoparticles [17,18], emulsions [19], and oil body [20]. The mechanism of this method is controlling the water solubility of lipophilic phenols via adjusting the pH values. At lower pH values, the lipophilic phenols showed poor water solubility. When the pH value was higher than the pKa of the hydroxyl group of phenols, the deprotonation of the hydroxyl group leads to an increase in the charge and water solubility of phenols. Then, the deprotonated/water-soluble phenols become protonated/water insoluble once the pH value is lower than the pKa. These protonated lipophilic phenols molecules show different behaviors in different systems: (1) In pure water systems, these molecules recrystallize into crystal nucleus, which grows into phenol crystal and precipitates [21]; (2) in the presence of biosurfactant or biopolymers, these amphiphilic molecules are absorbed onto the surface of the crystal nucleus and prevent its growth, and biosurfactant/biopolymer coated phenol nanoparticles are formed [15,17,22]; (3) in the presence of delivery systems with hydrophobic domains (such as liposomes, emulsions, and zein nanoparticles), the protonated lipophilic phenol molecules penetrate into hydrophobic domains of delivery systems [23]. Among these phenol loaded delivery systems fabricated by the pH-driven method, biosurfactant/biopolymer-stabilized phenol nanosuspensions showed grea<sup>t</sup> potential due to their easier procedure and higher loading capacity. While the nanosuspensions showed grea<sup>t</sup> advantages, they were mainly applied to encapsulate curcumin. Whether they could be applied to encapsulate other lipophilic phenols is still unknown. The influence of phenol properties on the formation and structure of phenol nanosuspensions needs to be explored.

Caseinate as the main source protein obtained from milk has been widely applied in food industries due to its desirable amphiphilic properties and abundant essential amino acids [24]. In previous research, caseinate has been employed concerning hydrophobic nutrients, such as curcumin, beta-carotene [25], and quercetin, which could improve their stability and water solubility. Compared with some other synthetic compounds, for instance, inorganic materials and small molecule surfactants, caseinate as a kind of natural material is more easily accepted by consumers and food factories. Our previous research indicated that caseinate is a wonderful encapsulation material for lipophilic nutrients via the pH-driven method with the highest encapsulation efficiency and loading capacity compared with serval kinds of proteins and polysaccharides [17]. All of this information proved the potential of caseinate as shell and encapsulation compounds to construct nanoparticles with thymol. However, the relevant results and information of nanoparticle fabrication with thymol and caseinate via the pH-driven method still need to be reported.

Therefore, the main objective of this study was to fabricate thymol nanosuspensions stabilized by caseinate via the pH-driven method and to check if the pH-driven method is suitable for thymol nanosuspensions. First, the chemical stability of thymol in different pH value conditions was evaluated. Then, the caseinate-stabilized nanosuspensions were prepared through the pH-driven method, and the stability, encapsulation efficiency, and particle properties were determined. Finally, the antibacterial properties of encapsulated thymol were also investigated. These results will provide important information for the encapsulation of thymol via the pH-driven method, which is useful for application in the fabrication and design of food cosmetics and pharmaceutical products.
