Antimicrobial efficacy of Thymbra capitata (L.) Cav. essential oil loaded in self-assembled zein nanoparticles in combination with heat

https://doi.org/10.1016/j.indcrop.2019.03.003Get rights and content

Highlights

  • Carvacrol was identified as the major compound of Thymbra capitata oil (TCEO).

  • Zein, a plant protein isolated from maize, is proposed as biocompatible carrier.

  • Novel self-assembled zein nanocapsules loaded with TCEO (zn-TCEO) are described.

  • Valuable synergistic effects were obtained when combining mild heat and zn-TCEO.

  • The use of zn-TCEO might improve product higienization or surface disinfection.

Abstract

This study reports on the chemical composition of Thymbra capitata essential oil (TCEO) and its antimicrobial activity when applied with heat either as a suspension (s-TCEO) or loaded in self-assembled zein nanoparticles (zn-TCEO). Zein, a plant protein isolated from corn and maize, is proposed as natural, and biocompatible carrier. TCEO composition was analysed by GC–MS, and 35 components were identified. Carvacrol, a monoterpenoid, was the major constituent (73.8%). zn-TCEO were prepared under low shear conditions and characterized according to droplet size (<180 nm) and encapsulation efficiency (77.8%). The two TCEO formulations (s-TCEO and zn-TCEO) were compared in terms of antibacterial activity against Escherichia coli O157:H7 Sakai and Listeria monocytogenes EGD-e. The zn-TCEO displayed a greater bacteriostatic activity than s-TCEO, probably due to their improved dispersion in the growth media. However, zn-TCEO exerted a lower bactericidal activity than s-TCEO, probably due to the EO progressive release. The combination of TCEO and heat (53 °C) exerted valuable synergistic lethal effects, causing the death of up to 5 log10 cycles of both microorganisms. The effectiveness of zn-TCEO was especially improved at pH 4.0. Therefore, the application of this new delivery system, designed to encapsulate and protect EOs, and ensure their controlled release, might represent an advantageous alternative for food, cosmetic or pharmaceutical industries to improve the efficacy of higienization processes or surface cleaning and disinfection procedures when combined with mild heat.

Introduction

Plant essential oils (EOs) are natural compounds extracted mainly from aromatic and medicinal plants. Due to their antimicrobial properties, they have been frequently recommended as biocides, preservatives or for cleaning and disinfection procedures (Burt, 2004; Souza et al., 2016). However, their chemical instability due to oxidation, high reactivity, and hydrophobicity thwarts any attempt to incorporate them directly into cleaning solutions, cosmetic products, or food beverages (Donsì and Ferrari, 2016). New delivery systems have therefore been designed in order to protect their chemical properties and ensure their controlled release (Prakash et al., 2018).

Active encapsulation has mainly been used to protect bioactive compounds from adverse environmental factors, to enhance solubility of poorly soluble actives and to grant them certain specific properties, such as sustained or controlled release. Unfortunately, most of these promising nanoencapsulation methods require expensive equipment and present difficulties in the scale-up phase (spray drying, freeze-drying, etc.). Recent developments in the preparation of nanoparticles have been marked by a series of emerging issues: the requirement for less toxic reagents, a simplification of the procedure with the purpose of allowing economic scale-up, and optimization to improve yield and entrapment efficacy (Reis et al., 2006).

Thymbra capitata (L.) Cav. (syn. Thymus capitatus (L.) Cav., Lamiaceae) is an aromatic herb that grows in the Mediterranean area and produces an essential oil (TCEO). Thanks to its recognized antimicrobial and antioxidant properties, it can be used as a preservative for foodstuffs and formulations (Delgado-Adámez et al., 2017; Falcó et al., 2018; Neves et al., 2017).

Several natural polymers such as zein (Weissmueller et al., 2016), casein (Peñalva et al., 2018), or chitosan (Yuan et al., 2016) have been proposed as natural, and biocompatible carriers for different actives. Zein is a plant protein isolated from corn or maize belonging to a family of prolamines which are composed of hydrophobic aminoacids (Salman et al., 2013). In this context, the use of zein in conjunction with a controlled self-assembly method for encapsulation of TCEO would allow us to develop a carrier for this natural, lowcost, biocompatible and GRAS (generally recognized as safe) EO (Patel and Velikov, 2014).

Most of the studies that propose the encapsulation of EOs describe how their progressive release from nanocapsules can help inhibit microbial growth by developing a prolonged bacteriostatic activity over time (Chouhan et al., 2017). However, when a bactericidal effect is required in a short treatment, as tends to occur during higienization or surface cleaning and disinfection, the control of the liberation of EOs might entail a limitation, since the doses required to exert a bactericidal effect are not easily achieved.

In this regard, remarkable synergistic lethal effects have been described when EO suspensions (Espina et al., 2010; Guevara et al., 2015; Raybaudi-Massilia et al., 2009) or nanoemulsions (de Carvalho et al., 2018; Mate et al., 2016; Pagán et al., 2018) are applied in combination with physical processes, for instance mild heat treatments (50–60 °C). Such combined treatments have been proposed not only as alternatives to traditional fruit juice preservation methods, but also as a good means of eradicating biofilms of Listeria monocytogenes, Escherichia coli and Staphylococcus aureus from plastic surfaces (Espina et al., 2017). The described synergism has enabled the reduction of treatment temperatures and/or antimicrobial doses: in view of the strong flavor of most EOs, this can represent an enormous advantage.

As far as we can ascertain, the behavior of zein nanocapsules under heat treatments or of the antimicrobial properties of encapsulated EOs with zeins in combination with heat against pathogenic microorganisms has not been previously studied.

This research was therefore carried out in order to (i) evaluate the chemical composition of TCEO and its effectiveness in vitro on growth and survival of two pathogenic bacteria: E. coli O157:H7 Sakai and L. monocytogenes EGD-e; ii) obtain and characterize novel self-assembled zein nanocapsules loaded with TCEO (zn-TCEO) for TCEO protection and progressive release, and iii) to assess the antimicrobial efficacy of zn-TCEO as a single hurdle or in combination with heat as a function of treatment medium pH.

Section snippets

Thymbra capitata essential oil (TCEO)

One-hundred-percent pure and natural TCEO was obtained by hydrodistillation of the flower heads of Thymbra capitata. TCEO was kindly provided by the TELIC Group (Barcelona, Spain). Once received, the EO was kept at room temperature, in the dark, in sealed glass vials until used.

Following the method described by Friedman et al. (2002), we applied a vigorous shaking procedure to prepare TCEO suspensions (s-TCEO) in McIlvaine buffer at pH 7.0 and at pH 4.0.

Chemical analysis of Thymbra capitata essential oil

α-Pinene, camphene, β-pinene,

Chemical composition of Thymbra capitata essential oil (TCEO)

Qualitative and quantitative analysis of the TCEO is summarized in Table 1. Thirty-five volatile components were identified, representing 98.6% of all detected constituents. The components were grouped into main four classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes.

As shown in Table 1, TCEO contained mostly oxygenated monoterpenes, which accounted for 77.5% of its composition. Carvacrol was the component present in the

Conclusions

The use of self-assembled zein nanoparticles to encapsulate EOs might represent an alternative preferable to the use of EOs in suspension, not only in order to overcome their high volatility and to improve EO dispersion in hydrophilic solutions, but also in order to enhance their antimicrobial activity. Moreover, the use of a non-toxic, biocompatible, natural polymer isolated from corn or maize (zein) for EO nanoencapsulation, combined with a method that is simple and easy to scale up, permits

Acknowledgements

This study was financially supported by MINECO (Spanish Ministry of Economy and Competitiveness, Project No. AGL2015-69565-P), by the European project SKHINCAPS – “SKin Healthcare by Innovative NanoCAPsuleS” (H2020 - 685909), by FEDER, by the European Social Fund, and by the Aragonese Office of Science, Technology and University Research.

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These authors contributed equally to the work.

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