2.3. Preparation of Double W/O/W Emulsion

Double emulsions were prepared according to the multiple (double) emulsion–melt dispersion technique as summarized in Figure 1. Firstly, preheated water phase (liquid gentian extract, sodium alginate, and sodium chloride) was added dropwise to the melted lipid phase (solid lipid: Gelucire^® 43/01 or Gelucire^® 39/01 and lipophilic emulsifier) according to the composition represented in Table 1, with constant stirring on a magnetic stirrer at 500 rpm (IKA^® RCT standard, Staufen, Germany) at temperature (5–10 °C) above the lipid melting point. In the preliminary study, primary emulsion (W/O) was formulated with Span^® 80 or PGPR as lipophilic emulsifiers. The obtained primary emulsion (W/O) was homogenized at 15,000 rpm for 3 min by using a high-shear homogenizer (Ultra-Turrax, IKA^®, Staufen, Germany), while the temperature was constantly in the range presented in Table 1.

Process scheme.

A–D: The primary (W/O) emulsion composition.

* Emulsions were prepared at 44–49 °C; ** Emulsions were prepared at 48–53 °C; *** Span^® 80 or polyglycerol polyricinoleate.

In the second step, hot (temperature indicated in Table 1) primary emulsion (W/O) with PGPR as lipophilic emulsifier was dispersed in the outer water phase containing a hydrophilic emulsifier, sodium alginate, sodium chloride, trehalose with or without Sylysia^® 350 (Table 2). The temperatures of emulsions with Gelucire^® 39/01 as solid lipid in the primary emulsions were in the range 44–49 °C, whereas the temperatures of emulsions with Gelucire^® 43/01 were in the range 48–53 °C. In the preliminary study, Tween^® 80, Tween^® 20, or lecithin were used as hydrophilic emulsifiers. Trehalose was used in all formulations as a cryoprotectant. Subsequently, emulsion (W/O/W) was homogenized at 3000 rpm for 4.5 min by a high-shear homogenizer (Ultra-Turrax, IKA^®, Staufen, Germany). Furthermore, double emulsion (W/O/W) was continually stirred by a laboratory mixer (Heidolph RZR 2020, Heidolph Elektro GmbH & Co., KG, Kelheim, Germany) until the temperature of the prepared W/O/W emulsion was approximately 25 °C.

A–D: Double (W/O/W) emulsion composition.

* Corresponding primary emulsion was marked with the same letter in Table 1; PGPR-polyglycerol polyricinoleate. ** Tween^® 80, Tween^® 20, or lecithin.