Elsevier

LWT

Volume 127, June 2020, 109382
LWT

Thymol and carvacrol in nanoliposomes: Characterization and a comparison with free counterparts against planktonic and glass-adhered Salmonella

https://doi.org/10.1016/j.lwt.2020.109382Get rights and content

Highlights

  • Thymol and/or carvacrol as natural disinfectants for glass surfaces.

  • Free versus encapsulated thymol and/or carvacrol against a Salmonella pool.

  • Planktonic and sessile Salmonella effectively reduced by free thymol and/or carvacrol.

  • Brief 1-min treatments with free antimicrobials eradicated glass-adhered Salmonella.

  • Antimicrobial-loaded nanoliposomes displayed decreased anti-Salmonella activities.

Abstract

Nanoliposome-encapsulated and free thymol, carvacrol, and thymol/carvacrol (1:1) were assessed as antimicrobials against a Salmonella pool. Liposome-encapsulated thymol (TL), carvacrol (CL) and thymol/carvacrol (TCL) presented 230–270 nm diameter, satisfactory polydispersity (0.24–0.31), zeta potential of approximately −30 mV and high encapsulation efficiencies (~99%), remaining stable during 28 days at 4 °C. Minimum inhibitory concentrations (MIC) of free antimicrobials were 331 μg/mL. Encapsulation increased MIC to 663 (TL, CL) and 1325 μg/mL (TCL). Time-kill curves with antimicrobial preparations (265.0 μg/mL) demonstrated superior activities for free antimicrobials; among nanoliposomes, TCL exhibited higher activity. At MIC, antimicrobials impeded bacterial adhesion to glass. Without antimicrobials, Salmonella (~8 log Colony Forming Units (CFU)/mL) adhered to glass reaching 5.89 log CFU/cm2 after 15 min. Inactivation of glass-attached Salmonella was complete following 1-min treatments with free (MIC, 2 × MIC) or encapsulated (2 × MIC) antimicrobials. At MIC, antimicrobial-loaded nanoliposomes decreased adhered populations by ~3.9 log CFU/cm2; at 0.5 × MIC, reductions of 1.93 (CL) and 1.58 log CFU/cm2 (TCL) were observed. Free antimicrobials were more effective against planktonic and sessile Salmonella at contact times employed in food processing environments. Nanoliposome-encapsulated natural antimicrobials warrant investigations on surface disinfection schemes and incorporation into foods.

Introduction

Foodborne illnesses are commonly related to biological contaminants and Salmonella is among the main causes of outbreaks (EFSA, 2018; Tondo & Ritter, 2012). Bacterial adhesion and biofilm formation onto food-contact surfaces are important for cross-contamination and recontamination in food industries and services. Properties of materials and microbial cells define, largely, the capacity of bacteria to attach to surfaces and develop biofilms (Srey, Jahid, & Ha, 2013). Glass is a hydrophilic material widely used in food processing environments, even replacing stainless steel, for diverse applications due to its smoother surface and increased corrosion resistance (Carrasco, Morales-Rueda, & García-Gimeno, 2012; Moraes et al., 2019). However, Salmonella usually adhere and form biofilms on glass surfaces (Chia, Goulter, McMeekin, Dykes, & Fegan, 2009).

Prevention of microbial growth and reduction of populations to its lowest levels in food-contact surfaces relies on cleaning and sanitization. Chlorine-based chemicals, peroxygens and quaternary ammonium compounds are major disinfectants. However, loss of antimicrobial activity by interfering substances, generation of unwanted by-products and emergence of resistant bacteria are relevant issues. Additionally, the diminished susceptibility of biofilms to current disinfection strategies indicates the pertinence of novel approaches to eradicate bacteria (Coughlan, Cotter, Hill, & Alvarez-Ordóñez, 2016; Møretrø, Heir, Nesse, Vestby, & Langsrud, 2012).

There is a positive perception of consumers regarding the use of natural compounds as compared to chemical disinfectants (Galié, García-Gutiérrez, Miguélez, Villar, & Lombó, 2018). Specifically, essential oils (EOs), oily liquids extracted from plant materials, possess antimicrobial activities (Calo, Crandall, O'Bryan, & Ricke, 2015; Falcó, Verdeguer, Aznar, Sánchez, & Randazzo, 2019). EOs from thyme and oregano and their major constituents, thymol and carvacrol, are effective against broad microbial groups (Du et al., 2015; García-Salinas, Elizondo-Castillo, Arruebo, Mendoza, & Irusta, 2018; Čabarkapa et al., 2019).

However, EO compounds present low stability, high hydrophobicity and volatility. Encapsulation of EO constituents into proper carriers might circumvent these potential problems. Particularly, liposomes are colloidal structures composed of lipid bilayers and an aqueous inner core that can encapsulate lipophilic, hydrophilic and amphiphilic compounds. From such versatility, liposomes exhibit diverse applications in the food sector, such as the delivery of EO antimicrobials in foods and packaging materials (Calo et al., 2015; Emami, Azadmard-Damirchi, Peighambardoust, Valizadeh, & Hesari, 2016).

Although free EO constituents are increasingly acknowledged as efficient surface disinfectants, the effects of encapsulation are less explored (Merino et al., 2019). Actually, liposomal encapsulation might either enhance (Khosravi-Darani, Khoosfi, & Hosseini, 2016) or decrease (Peng et al., 2015) their antimicrobial activities. Considering that no studies were found showing the effects of thymol and/or carvacrol towards glass-adhered Salmonella, this investigation aimed to encapsulate thymol and/or carvacrol in liposomes, to characterize obtained liposomes, and to evaluate their antimicrobial action against planktonic and glass-adhered Salmonella as compared to free counterparts.

Section snippets

Microorganisms

A bacterial pool composed by three Salmonella enterica strains (S. Enteritidis SE86, from cabbage involved in food outbreak; S. Heidelberg, from poultry products; and S. Typhimurium, from swine feces) was used. Strains were cultured individually in Brain Heart Infusion broth (BHI; Merck, Germany) at 37 °C for 18–24 h. Optical density of cultures was adjusted to ~0.5 units at 630 nm using sterile BHI, corresponding to 8 log Colony Forming Units (CFU)/mL, as confirmed by plate counts in Xylose

Characterization of liposomes, EE and storage stability

Immediately after encapsulation, TL, CL and TCL exhibited similar sizes (230.9–267.7 nm), PDI (0.24–0.31), zeta potentials (−30.3 to −31.2 mV), with remarkable EE (~99%) (Table 1A). Properties of some nanoliposomal preparations encapsulating EO or EO constituents are compiled in Table 1B. Liposomes’ properties depend on the type of phospholipids, addition of other compounds (e.g. cholesterol), preparation method and the nature of encapsulated EO or constituent (Table 1). The PDI values of

Conclusion

Free thymol and/or carvacrol exhibited remarkable antibacterial activities against a 3-strain Salmonella pool. At MIC, antimicrobials were effective towards planktonic and sessile bacteria. Since a short-term contact (1 min) was sufficient to inactivate glass-adhered Salmonella (5.89 log CFU/cm2), these antimicrobials are alternatives to conventional disinfectants.

Antimicrobials were successfully encapsulated. Nanoliposomes presented adequate uniformity and high EE, remaining stable for 28 days

CRediT authorship contribution statement

Caroline Heckler: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft. Caroline Marques Maders Silva: Methodology, Investigation. Fabiola Ayres Cacciatore: Conceptualization, Methodology, Investigation. Daniel Joner Daroit: Formal analysis, Writing - original draft, Writing - review & editing. Patrícia da Silva Malheiros: Conceptualization, Resources, Supervision, Project administration, Funding acquisition, Writing - original draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for financial support.

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