Elsevier

Food Chemistry

Volume 197, Part A, 15 April 2016, Pages 75-83
Food Chemistry

Bactericidal action mechanism of negatively charged food grade clove oil nanoemulsions

https://doi.org/10.1016/j.foodchem.2015.10.015Get rights and content

Highlights

Abstract

Clove oil (CO) anionic nanoemulsions were prepared with varying ratios of CO to canola oil (CA), emulsified and stabilized with purity gum ultra (PGU), a newly developed succinylated waxy maize starch. Interfacial tension measurements showed that CO acted as a co-surfactant and there was a gradual decrease in interfacial tension which favored the formation of small droplet sizes on homogenization until a critical limit (5:5% v/v CO:CA) was reached. Antimicrobial activity of the negatively charged CO nanoemulsion was determined against Gram positive GPB (Listeria monocytogenes and Staphylococcus aureus) and Gram negative GNB (Escherichia coli) bacterial strains using minimum inhibitory concentration (MIC) and a time kill dynamic method. Negatively charged PGU emulsified CO nanoemulsion showed prolonged antibacterial activities against Gram positive bacterial strains. We concluded that negatively charged CO nanoemulsion droplets self-assemble with GPB cell membrane, and facilitated interaction with cellular components of bacteria. Moreover, no electrostatic interaction existed between negatively charged droplets and the GPB membrane.

Introduction

The essential oils are a diverse group of natural aromatic compounds isolated mostly from non woody plant materials (Edris, 2007). They possess terpenoids, especially monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), along with a variety of aliphatic hydrocarbons (low molecular weight), acids, alcohols, aldehydes and esters. Essential oils posses strong antibacterial and antifungal activities (Dorman and Deans, 2000, Jones, 1996, LisBalchin and Deans, 1997). Clove (Syzygium aromaticum L.) is an important medicinal plant, widely used in pharmaceutical and food industries. The main constituents of clove essential oil (CO) are eugenol, beta-caryophyllene, alpha-humelene and eugenyl acetate (Moon, Kim, & Cha, 2011) and have analgesic, anti-inflammatory and antimicrobial properties (Chaieb et al., 2007). For example, Joseph and Sujatha (2011) confirmed significant inhibitory activity of CO against Staphylococcus spp. with a minimum inhibitory concentration (MIC) value of 2.5% v/v.

Similarly Babu, Sundari, Indunmathi, Srujan, and Sravanthi (2011) compared antimicrobial efficacy of CO, garlic and cinnamon oils against Staphylococcus spp., Escherichia coli and Listeria monocytogenes. They confirmed more bacterial growth inhibition by CO & cinnamon oil than garlic oil. Mytle, Anderson, Doyle, and Smith (2006) studied the antilisteric activity of CO (1% and 2% v/v) on chicken frankfurters during cold storage and observed a significant decrease in L. monocytogenes contamination when stored at 5 and 15 °C. The above mentioned studies confirmed bacterial growth reduction potential of CO against food borne pathogens but, due to low water solubility and high volatility the antimicrobial effect was achieved at a high MIC value. It is of no doubt that essential oils possess bactericidal action against a variety of pathogenic microorganisms, but their action involves various mechanisms in the cell. The possible mechanisms for essential oils and their components against bacterial cells reported in the literature are (1) degradation of the cell wall, (2) damage to cytoplasmic membrane, (3) damage to membrane proteins, (4) leakage of cell contents, and (5) coagulation of cytoplasm and depletion of the proton motive force (Burt, 2004). However, the hydrophobicity of essential oils may reduce interaction with the bacteria in an aqueous environment, delays its bactericidal action and reduce its toxicity to bacteria (Burt and Reinders, 2003, Ultee et al., 2002). Few approaches have been proposed that can decrease the concentration of essential oils and reduce their sensory effects.

Encapsulation of essential oils may increase solubility and dispersibility in aqueous media, reduce adverse interaction with food components, and improve antimicrobial activity by promoting contact with bacteria. Common lipid encapsulation systems are emulsions (Rodriguez-Rojo, Varona, Nunez, & Cocero, 2012), microencapsulation (Solomon, Sahle, Gebre-Mariam, Ares, & Neubert, 2012) and liposomes (Mekkerdchoo, Patipasena, & Borompichaichartkul, 2009) where lipids are encapsulated by surfactants. Improved antimicrobial activities of clove oil, eugenol, oregano essential oil and cinnamaldehyde have already been reported when incorporated into encapsulated matrices (Arana-sanchez et al., 2010, Hill et al., 2013, Shah et al., 2013). Liang et al. (2012) prepared a peppermint oil (PO) loaded nanoemulsion based delivery system with a particle size <200 nm and confirmed a prolonged antimicrobial activity of PO nanoemulsion compared to PO. The MIC values of both PO and PO nanoemulsion were the same i.e., 0.5% v/v. In the case of L. monocytogenes the bacterial number reached 104 CFU/ml after 36 h, while it went up to 107 CFU/ml when treated with PO. They observed long term inhibition of bacterial growth after treatment with a PO nanoemulsion even though both the PO and PO nanoemulsion had the same MIC values. Similarly, carvacrol, limonene and cinnamaldehyde based delivery systems stabilized by lecithin, pea protein, sugar ester and a combination of Tween 20 and glycerol monooleate showed enhanced antimicrobial activity against E. coli and Lactobacillus delbrueckii. The antimicrobial formulations stabilized with sugar ester and a combination of Tween 20 and glycerol monooleate inhibited bacterial growth up to 101 CFU/ml within 2 h and completely inactivated it after 24 h (Donsi, Annunziata, Vincensi, & Ferrari, 2012).

Nanoemulsions are commercially valuable delivery systems because they have the unique characteristics of small size and high surface area, optical clarity, and reduced rate of gravitational separation and flocculation. However, droplet size of nanoemulsions may change by Ostwald ripening (growth of larger droplets at the expense of small droplets) (Kabalnov, 2001). Donsi et al. (2012) overcome Ostwald ripening of carvacrol, d-Limonene and trans-cinnamaldehyde nanoemulsions by the addition of sunflower oil into the lipid phase that resulted in stable nanoemulsions.

In addition to Ostwald ripening inhibition, stable nanoemulsion preparation involves surfactants that lower surface tension between the oil and water phase. A variety of food grade gums, casein and succinylated starch (modified) based surfactants have been used by a variety of researchers for the preparation of nanoemulsions. However, simple starches have not been utilized because of their large size and predominant hydrophilic characteristics. Recently, researchers prepared an emulsion based microcapsules of thymol and carvacrol by combining Tween 20 and gum arabic as the surfactant, and determined their antimicrobial activity against pathogenic fungi (Aspergillus niger) and food borne microbes Staphylococcus aureus, E. coli, Listeria innocua and Saccharomyces cerevisiae. Thymol microcapsules inhibited the growth of bacteria and molds at an MIC value of 250 ppm. Whereas, carvacrol showed inhibition at a lower MIC value of 225 ppm (Guarda, Rubilar, Miltz, & Galotto, 2011). Therefore, clove oil was selected as the antimicrobial agent in a nanoemulsion stabilized by purity gum ultra (PGU), which is a new food grade, succinylated waxy maize starch designed to emulsify lipids and was selected as the surfactant. PGU produced stable, small droplets (254 nm) of orange oil in water emulsions at a low surfactant ratio i.e., 1% wt/wt, while with gum Arabic the droplet diameter was (497 nm) at a high surfactant ratio of 5% wt/wt (Mao et al., 2009). PGU may be a suitable candidate to replace the synthetic surfactants used in food industries because of its efficiency at lower concentrations and consumer friendly label (Qian, Decker, Xiao, & McClements, 2011a). The objective of our study was to use PGU as an alternative to a synthetic surfactant (Tween 80) and to compare their structural properties in relation to antimicrobial activity against Gram positive (L. monocytogenes, S. aureus) and Gram negative (E. coli) bacterial strains.

Section snippets

Materials

Clove oil (S. aromaticum L.), extracted by supercritical fluid extraction method, was purchased from Jishui County Man Herbal Medicinal Oil Refinery Co., Ltd. (Jiangxi, China). Canola oil was purchased from a local market and used without further purification. Purity gum ultra (PGU), a succinylated waxy maize starch, was purchased from National Starch (Bridgewater, NJ, USA). The bacterial strains L. monocytogenes ATCC19114, S. aureus ATCC 25923 were purchased from Haibo Biotechnology Co. Ltd,

Formation of clove oil nanoemulsions

To get a stable essential oil nanoemulsion it is necessary to blend it with an additional organic phase. For example, Donsi et al. (2012) prepared a stable lecithin based nanoemulsion by blending d-limonene with palm oil (1:1). Similarly, other researchers have also reported the formation of an essential oil nanoemulsion by blending with medium chain triglyceride (MCT) or long chain triglyceride oil and this formed emulsions that were stable for a longer time period. Therefore, in our study we

Acknowledgements

This work was financially supported by National 863 Program 2011BAD23B02, 2013AA102207, NSFC – China, 31171686, 30901000, 111 project-B07029 and PCSIRT0627.

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