Ethylcellulose oleogel as a prolonged release delivery system for probiotics. Development and validation of a cold-mixing method.

Abstract

Ethylcellulose thickened oil (EC oleogels) has a significant retarding effect on lipolysis and may be used for controlled-release formulations. However, the high temperature (above 150°C) used during the gel-forming process becomes an obvious limitation that hinders the application of EC oleogels to temperature-sensitive active ingredients. A cold-mixing procedure that could allow for an encapsulation at a temperature of around 40°C is proposed in this study. A warm solution of EC in oil is allowed to cool down, and just before the gelation, the solution is mixed with a cool oil dispersion of the heat-sensitive ingredient. The temperature drops, and the gel is solidifying within a few minutes. The evaluation shows that the oleogels formed have a lower hardness and oil entrapment than the hot-mixed EC oleogels. However, the gel character remains, displaying a comparable digestion resistance. There is a limited loss of viability of the probiotics when encapsulated in the oleogels, about 50%. The loss of viability when released from a cold-mixed oleogel under digestive conditions becomes about 90% due to the detrimental digestive environment. This observation can be compared to 99% loss when released from oil under digestive conditions. Thus, the in vitro evaluation of an EC oleogel suggests that systems formed after cold-mixing can provide prolonged delivery of oxygen and bile-sensitive bioactive ingredients without exposing them to detrimental temperatures during the formulation.

Highlights

Introduction

Probiotics are sensitive to acidity, enzymes, and bile salts, which are present in high amounts in the duodenum and jejunum (de Vos et al., 2010; Konstantinov et al., 2008). During storage, they are also sensitive to humidity and oxygen in the surrounding environment. Encapsulation and controlled-release formulations have been proposed to handle this high sensitivity of probiotics (Borgogna et al., 2010; Burgain et al., 2011). Investigated materials used for encapsulation of probiotics include different alginates (Krasaekoopt et al., 2003; Rowley et al., 1999), gellan gum, xanthan gum (Sultana et al., 2000; Sun & Griffiths, 2000), chitosan (Chávarri et al., 2010; Mortazavian et al., 2008), and starch (Sajilata et al., 2006). Also, water in oil emulsions has been proposed as microbiological encapsulation matrixes for oral delivery (Zhuang et al., 2021).

The encapsulation systems intend to create physical barriers reducing direct contact between the probiotics and the adverse environments in the gastrointestinal system. It is also essential that the encapsulation system allows for a release of the probiotics in a viable and metabolically active state at the target location (Picot & Lacroix, 2004).
Ethylcellulose, EC, is the product of etherification between cellulose and ethyl chloride or sulfate in a highly alkaline solution (Koch, 1937). EC is non-toxic, has undergone clinical evaluation, and received approval as an excipient in many formulations for both adults and children (Rowe et al., 2009; Wasilewska & Winnicka, 2019). It is often used as dried films in pharmaceutics to create a controlled release of tablets (Arora & Mukherjee, 2002; Heng et al., 2003; Melzer et al., 2003; Pena Romero et al., 1991; Wu et al., 2003).

Ethylcellulose (EC) can form stiff gels when dissolved in triglyceride oils. These materials are usually called oleogels (Heng et al., 2005; Kolpak & Blackwell, 1976; Laredo et al., 2011). The EC/triglyceride oleogels are formed after dissolving the softened and melted EC in the oil using a heating procedure (the solubilization temperature is just above Tg, around 130-140°C) followed by a cooling to the gelation temperature (Davidovich-Pinhas et al., 2014). The gelation temperature is typically between 50 and 100°C. (Davidovich-Pinhas et al., 2014; Davidovich-Pinhas et al., 2015b).

The key findings from the literature on the physical characterization (i.e., rheological properties, mechanical properties, and thermal properties) of EC oleogels are as follows:
Ⅰ) Increases in the EC concentration or molecular mass lead to higher strength, viscosity, and hardness of the oleogels. (Davidovich-Pinhas et al., 2015c; Gravelle et al., 2013; Gravelle et al., 2016; Heng et al., 2005; Zetzl et al., 2012).

Ⅱ) Parameters related to the gelation process, such as cooling rate and gelation temperature, induce structural changes that influence the strength of the gels (Davidovich-Pinhas et al., 2015c).
The EC oleogels can be quite stiff, typically with an elastic modulus (G’) of the gel of about 100 kPa for an oleogel with 11% EC at 60°C. (Davidovich Pinha 2015b).

Davidovich Pinha and co-workers have studied the structure of EC before solubilization and found that the original structure is mainly amorphic with local crystallites. When heated over the Tg (glass transition temperature), the amorphous structure softens, and the crystallites melt (Davidovich-Pinhas et al., 2014). They also discuss the structure of the solution above and the gel below the gelation temperature in their papers. They concluded that the gel is three-dimensional, they suggest that there are junction zones, that there are microgels present at temperatures just above the gelation temperature, and that the gelation does not involve any change on an organized secondary structure level (Davidovich-Pinhas et al., 2014, Davidovich-Pinhas et al., 2015b).

The most commonly proposed application of EC oleogel is a non-digestible or slowly digestible fat substitute (Adili et al., 2020). Meanwhile, EC oleogels have been proposed as controlled delivery formulations for sensitive hydrophobic active ingredients (O’Sullivan et al., 2016). In our recent study, where various oil-based encapsulation systems for probiotics were assessed, the results indicate that EC oleogels exhibit particularly promising capabilities in retarding lipolysis and provide protection against bile salts being present in the duodenum (Zhang et al., 2022). However, the high temperature needed to form the oleogel is a major challenge for the applications of EC oleogels to encapsulate temperature-sensitive biologics.

This paper evaluates the possibilities of applying EC oleogels as an encapsulating and prolonged release system for probiotics. This result has been obtained by developing a cold-mixing method that allows the bacteria to be introduced into the oleogel at a non-critical temperature (t<40 °C). The method is based on the gradual character of the solidification when the EC solution in a triglyceride is cooled down from the solubilization temperature (>140°C) down to a temperature when they start to solidify (between 40°C and 100°C, depending on concentration, molar mass, and time).

The properties of the cold-mixed oleogels, such as hardness, oil entrapment capacity, and digestibility, were evaluated. The viability of the probiotics was evaluated before and after encapsulation in the EC cold-mixed oleogels to examine if the formulation itself was detrimental to the viability of the probiotics. The release of probiotics was evaluated using in vitro lipolysis. The demonstration organism is Limosilactobacillus reuteri.

Materials

The investigated ethylcellulose were described according to their viscosities in a 5% solution in toluene/ethanol 80:20. The EC samples were 22 cP, 46 cP, 100 cP, and 300 cP, respectively, according to the supplier. The extent of ethylation was 48%. The EC samples were supplied by Sigma-Aldrich (St. Louis, MO, USA). As the oil component for the oleogel, a medium-chain triglyceride oil (MCT) was chosen.

Read more

Lingping Zhang, Marie Wahlgren, Elin Oscarsson, Björn Bergenståhl, Ethylcellulose oleogel as a prolonged release delivery system for probiotics. Development and validation of a cold-mixing method., Food Hydrocolloids, 2025, 111339, ISSN 0268-005X,
https://doi.org/10.1016/j.foodhyd.2025.111339.