Bactericidal action mechanism of negatively charged food grade clove oil nanoemulsions

Food Chemistry 197 (2016) 75–83

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Bactericidal action mechanism of negatively charged food grade clove oil nanoemulsions Hamid Majeed a, Fei Liu a, Joseph Hategekimana a, Hafiz Rizwan Sharif a, Jing Qi a, Barkat Ali a, Yuan-Yuan Bian a, Jianguo Ma a, Wallace Yokoyama b, Fang Zhong a,⇑ a b

Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China Western Regional Research Center, ARS, USDA, Albany, CA 94710, United States

a r t i c l e

i n f o

Article history: Received 20 May 2015 Received in revised form 23 August 2015 Accepted 5 October 2015 Available online 22 October 2015 Keywords: Clove oil Anionic Food grade nanoemulsion Antimicrobial Gram positive bacteria

a b s t r a c t 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. Ó 2015 Elsevier Ltd. All rights reserved.

1. 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 & Deans, 2000; Jones, 1996; LisBalchin & 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, antiinflammatory 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

⇑ Corresponding author. E-mail address: [email protected] (F. Zhong). http://dx.doi.org/10.1016/j.foodchem.2015.10.015 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

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 & Reinders, 2003; Ultee, Bennik, & Moezelaar, 2002). Few approaches have been proposed that can decrease the concentration of essential oils and reduce their sensory effects.

76

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

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 (Aranasanchez et al., 2010; Hill, Gomes, & Taylor, 2013; Shah, Davidson, & Zhong, 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. 2. Materials and methods 2.1. 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, (Qingdao, China) and E. coli HB2151 was purchased from Distance Biotechnology Co. Ltd, (Hangzhou, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Interfacial tension measurements The water oil interfacial tension values were measured using a tensiometer (DCAT 21, DataPhysics Instruments, and Germany) with the Wilhelmy plate method (Hecht, Wagner, Landfester, & Schuchmann, 2011). The dimensions of the Pt–Ir plate were 10  19.9  0.2 mm3. Aqueous dispersions of PGU or Tween were prepared at concentrations of 1% and 2%. The interfacial tensions between the aqueous dispersions of surfactant and oil phases composed of mixtures of CA & CO were measured. All measurements were carried out in triplicate at 25 °C. The interfacial tension values are shown in Table 1.

Table 1 Interfacial tension (mN/m) measurements (n = 3) of an aqueous dispersion of PGU or Tween 80 and oil phases of various ratios of canola oil (CA) and clove oil (CO). 10% v/v CA 1% wt/wt PGU 2% wt/wt PGU 1% wt/wt Tween 2% wt/wt Tween 80

1:9 CO:CA

3:7 CO:CA

5:5 CO:CA

7:3 CO:CA

19.1 ± 0.02

15.7 ± 0.02

12.7 ± 0.01

10.2 ± 0.02

ND

18.0 ± 0.02

14.5 ± 0.009

9.8 ± 0.03

7.3 ± 0.02

ND

5.1 ± 0.02

2.9 ± 0.01

2.4 ± 0.01

0.8 ± 0.02

ND

4.2 ± 0.01

2.2 ± 0.02

2.0 ± 0.01

0.3 ± 0.01

ND

CO: Clove Oil, CA: Canola Oil, PGU: Purity Gum Ultra, ND: Not Determined, (n = 3).

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

2.3. Preparation of emulsions An aqueous solution of PGU 2% (w/w) was prepared by dispersing the dried powder in deionized water at room temperature and stirring overnight to enhance hydration of the starch prior to homogenization. Pure canola oil (CA, 10% v/v), and a mixture of CO:CA at ratios of 1:9, 3:7 and 5:5 (10% v/v) were used as core materials. Pure CA (without mixing with CO) nanoemulsion was prepared as a control. These mixed oil ratios were selected because above these ratios CO affects the nanoemulsion stability. CO was added with a Pasteur pipette in CA oil under continuous stirring at 250 rpm to ensure complete homogenization. During mixing tube was wrapped with aluminum foil and the processing temperature was kept at 25 °C. The oil and aqueous starch phases were premixed with a high-speed homogenizer (Ultra-Turrax T25 IKA Janke and Kunkle, GmbH and CO KG, Germany) at 13,500 rpm for 2 min at room temperature. These coarse emulsions were finely dispersed with a high pressure homogenizer (IKA-Labor Pilot 2000/4, IKA-Werke GmbH and Co. Staufen, Germany) at 50, 100, and 150 MPa for 1, 3, 5, 7, 10, 15 and 20 passes. During processing a heating exchanger was used to control the inlet, operational, and outlet temperatures at 15 °C. 2.4. Particle size distribution and zeta potential measurements Emulsion particle size and f potential were measured by dynamic light scattering and phase analysis light scattering (Zetasizer Nano ZS, Malvern Instruments, Malvern, U.K.), respectively, using 1 ml emulsion samples diluted 100 with deionized water to avoid multiple light scattering effects. Measurements were performed after 1, 15, and 30 days of storage. For dynamic light scattering, the particle size data are reported as Z-average mean diameter and polydispersity index (PDI). 2.5. Adhesion of nanoemulsion droplets and microorganisms Adhesion of nanoemulsion droplets to the Gram positive bacteria (GPB) L. monocytogenes and S. aureus and Gram negative bacterial (GNB) E. coli strains were evaluated using the procedure described by Dillen et al. (2008). Briefly, 2 ml of bacterial suspensions in PBS, nanoemulsions were added and mixed gently. Size of bacteria before and after exposure to the different nanoemulsion formulations was determined by Photon Correlation Spectroscopy using a Zetasizer 3000 (Malvern Instruments, Malvern, UK) as described above. 2.6. Antimicrobial assays 2.6.1. Determination of minimum inhibitory concentration (MIC) The agar dilution method was used to measure the antimicrobial activities of CO and CO nanoemulsions against GPB (L. monocytogenes and S. aureus) and GNB (E. coli) strains. The method described by Hammer, Carson, and Riley (1999) was used with a few modifications. Briefly, a series of dilutions 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.20%, 0.22%, 0.24%, 0.26%, 0.28% v/v of CO and CO:CA nanoemulsions were prepared in trypticase soy agar yeast extract (TSAYE) and LB agar. Plates were solidified at room temperature for 30 min prior to inoculations with 10 ll solution of each bacterium containing approximately 104 CFU/ml. Media alone and media with CA oil nanoemulsion without CO were used as controls. Inoculated plates were incubated at 37 ± 2 °C for 24 h. MICs against each strain were determined as the lowest concentration of CO and CO nanoemulsion inhibiting the visible growth of test microorganism on the agar plate. All experiments were performed in triplicate.

77

2.6.2. Time-kill studies The dynamic time-kill plots for GPB (L. monocytogenes, S. aureus) and GNB (E. coli) against CO and CO nanoemulsions at their MIC were determined. GPB strains were cultured in trypticase soy broth (TSAYE) and GNB in LB media, incubated at 37 °C under continuous agitation at 120 rpm for 10 h to obtain a stationary growth phase. The final concentration reached in the culture was 104–105 colony forming units/milliliter (CFU/ml). The bacterial culture was mixed with CO and CO nanoemulsions at their MIC values. The MIC values of CO and CO nanoemulsions against L. monocytogenes were 0.1% & 0.12% v/v. Whereas, for S. aureus and E. coli the MIC values were 0.14% & 0.16% v/v and 0.08% & 0.1% v/ v, respectively. Aliquots of 0.1 ml were taken from each sample after 0, 2, 4, 6, 8, 10, 12, 24, 36, 48 h and were serially diluted and spread in duplicate on TSAYE and LB agar plates. The plates were incubated at 37 ± 2 °C for 24 h. Colonies were counted manually and calculated by dilution times. All experiments were carried out in triplicate. 2.6.3. Fluorescence microscopy Fluorescence microscopy observations were carried out with an Eclipse TE2000S inverted microscope (Nikon), equipped with a B2A filter (excitation filter wavelengths: 450–490 nm, dichromatic mirror cut-on wavelength: 500 nm, barrier filter wavelengths: 515 nm cut-on), fitted with a high-pressure mercury burner as a light source. The images were acquired with a digital camera (DS-5M Digital Sight Camera System, Nikon), through a 20 lens (Nikon). Nile red (Sigma Aldrich, Germany) was used as a fluorescent lipophilic stain. It excites at 485 nm, and emits at 525 nm. The Nile Red was dissolved in ethanol at a concentration of 1 mg/ml; a sample of 100 ll of this solution was added to 1 ml of nanoemulsion to stain the oil droplets. Nanoemulsion with Nile Red was subsequently added to a culture medium containing 104 CFU/ml of Gram positive and Gram negative bacterial strains in a stationary phase at their MIC values. At a fixed time, a drop of the sample was mounted onto a glass slide, enclosed with a cover slit and observed. 3. Results and discussion 3.1. 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 blended CO with CA in different ratios. In order to understand the efficiency of surfactant we measured the interfacial tension of a CO and CA mixture with an emulsifier solution (Table 1). Increasing the CO: CA ratio resulted in a lower interfacial tension. The interfacial tension (mN/m) results suggest that eugenol (main component of CO) contributes to the surface activity, and CO is known to form small droplets, as reported in other studies (Hammer et al., 1999; Hecht et al., 2011; Qian et al., 2011a; Ru, Yu, & Huang, 2010). Smaller droplets decrease gravitational separation and favor emulsion stability (resist coalescence), therefore formation of the smallest droplet diameter was used to determine the proper homogenization pressure and processing cycles. Fig. 1 shows the effect of homogenization passes and variable pressures on droplet mean diameters for emulsions composed of a CO:CA oil mixture (5:5% v/v) stabilized by 2% wt/wt PGU, homogenized at 50, 100

78

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

450

Mean Diameter (nm)

400

50 MPa 100 MPa 150 MPa

350 300 250 200 150 100 0

5

10

15

20

Cycles Fig. 1. Effect of homogenization pressure and number of processing cycles on mean diameters (n = 3) of clove oil (CO) nanoemulsion containing 2% wt/wt PGU (purity gum ultra) and mixture of CO:CA (5:5% v/v).

and 150 MPa. With increasing pressure and number of passes the droplet size decreased significantly as reported in various studies (McClements & Rao, 2011; Qian, Decker, Xiao, & McClements, 2011b; Tan & Nakajima, 2005; Yuan, Gao, Zhao, & Mao, 2008). However with the current emulsion formulation (mixture of CO: CA, 5:5% v/v) oil and 2% (wt/wt PGU) droplet size decreased gradually from passes 1 to 10 at 50 and 100 Mpa, respectively (Fig. 1). After 10 passes the change in droplet size was negligible. On the other hand, droplet size decreased significantly from passes 1 to 5 at 150 MPa and further passes had no effect on size. We used these results to select pressure and homogenization passes in order to produce uniform droplet size for subsequent studies. Mean droplet diameter decreased from 400 to 225 nm and 275 to 170 nm when homogenized at 50 and 100 MPa, respectively. Correspondingly, polydispersity index decreased from 0.220 to 0.102 from 1 to 10 passes at 50 MPa and 0.148 to 0.136 at 100 MPa. The smallest droplet diameters (200–150 nm) and polydispersity indices (0.149–0.098) were produced by homogenization with 5 passes at 150 MPa and therefore these conditions were used to prepare CO:CA nanoemulsions in this study. The droplet size distribution of emulsions containing different concentrations of CO:CA when prepared at 150 MPa with 5 processing passes and 2% wt/wt PGU are shown in Table 2. However, in the case of Tween 80 we used 150 MPa and 2 passes to obtain

the same particle size as that of a 5:5% v/v CO:CA PGU nanoemulsion. The larger oil droplet diameter for 10% CA emulsion (without CO) is an indication that the phytophenols in CO may act as a cosurfactant and its role as a co-surfactant is supported by the interfacial tension data (Table 1). Up to a critical loading concentration of CO, the droplet diameter and PDI decreases. Surprisingly, at concentrations above a critical clove oil ratio i.e., 5:5% v/v, CO:CA droplet diameter distribution increases at the same homogenization pressure and number of processing cycles. Terjung, Loeffler, Gibis, Hinrichs, and Weiss (2012) observed similar effects of polyphenols and oil emulsions. They observed that critical limits for eugenol and carvacol were 50% and 30% wt/wt, respectively, in the oil phase. Above the critical limit the emulsions (7:3% and 9:1% v/v CO:CA) separated into a cream layer and serum layer after standing for several hours despite the smaller initial droplet sizes. The particles in the cream layer grew larger without flocculation but no coalescence was observed in emulsions that had a lower CO content. This phenomenon has been attributed to Ostwald ripening, in which the mean droplet size of an emulsion increases over time due to diffusion of molecules from small to large droplets (Terjung et al., 2012). In emulsions formed from a mixture of a water insoluble component and a component with higher water solubility, Ostwald ripening can be controlled by increasing the water insoluble component. Therefore, the loading of emulsion

Table 2 Properties of oil in water nanoemulsions prepared with either PGU or Tween 80 as a surfactant and with an oil phase of various ratios of canola oil and clove oil. S. no

Surfactant

Volume ratio of CA to CO

NPC

Particle size (nm) (Mean ± SD)

PDI (Mean ± SD)

Zeta potential (mV) (Mean ± SD)

1 2 3 4 5 6 7

PGU PGU PGU PGU PGU PGU Tween 80

10% CA 1:9% CO:CA 3:7% CO:CA 5:5% CO:CA 7:3% CO:CA 9:1% CO:CA 5:5% CO:CA

5 5 5 5 5 5 2

203.9 ± 0.96 188.4 ± 0.47 174.4 ± 0.37 150.6 ± 0.23 319.6 ± 0.28 425.9 ± 0.04 151.3 ± 0.56

0.20 ± 0.002 0.150 ± 0.003 0.13 ± 0.002 0.09 ± 0.0004 0.43 ± 0.002 0.36 ± 0.026 0.14 ± 0.002

32.7 ± 0.21 30.1 ± 0.23 30.3 ± 0.47 28.1 ± 0.23 36.5 ± 0.42 39.8 ± 0.23 Neutral

CA: Canola Oil, CO: Clove Oil, PGU: Purity Gum Ultra, PDI: Polydispersity Index, NPC: Number of processing cycles. Values are expressed as Mean ± S.D (n = 3).

79

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

Day 1 Day 15 Day 30

220

Mean Particle Diameter (nm)

200 180 160 140 120 100 80 60 40 20 0 1:9 CO:CA PGU

3:7 CO:CA PGU

5:5 CO:CA PGU

5:5 CO:CA TW

Fig. 2. Mean droplet diameter (n = 3) during one month storage at room temperature for PGU based nanoemulsions possessing 10% v/v oil phase prepared with different ratios of clove to canola oil at 1500 bar pressure with 5 processing cycles.

with CO can be limited by the amount and solubility properties of another lipid phase.

Table 3 The minimum inhibitory concentrations (MIC) of clove oil (CO) and nanoemulsions of CO for L. monocytogenes, S. aureus, and E. coli (n = 3).

3.2. Storage stability of clove oil nanoemulsions Storage tests were performed in order to determine the stability of emulsions and the mean droplet diameters. The effect of storage time on mean droplet diameter of nanoemulsions prepared with different ratios of CO:CA stored at room temperature for 1, 15 and 30 days are shown in Fig. 2. Droplet diameter increased 20–25 nm during 30 days of storage for all formulations. Liang et al. (2012) also observed that peppermint oil nanoemulsion droplet diameter changed by 20–30 nm over one month of storage at ambient conditions. No creaming or phase separation was observed. Therefore, four nanoemulsion formulations (1:9%, 3:7%, 5:5% v/v PGU and 5:5% v/v Tween 80) of CO:CA demonstrated a stable particle diameter during storage.

Antimicrobial component

Final quantity of CO and CO nanoemulsions added to aTSAYE and b LB (% v/v)

L. monocytogenes

CO PGUc CO Nanoemulsion Tween 80 CO Nanoemulsion

0.1 0.1

CO PGU CO Nanoemulsion Tween 80 CO Nanoemulsion

0.14 0.14

CO PGU CO Nanoemulsion Tween 80 CO Nanoemulsion

0.08 0.1

S. aureus

E. coli

3.3. Antimicrobial activity of clove oil nanoemulsion a

Composition of emulsified CO and CO was analyzed by GC–MS (see Supplementary Information for the method). The concentration of eugenol, the major component, decreased by 5% (75–70%), otherwise the composition of CO nanoemulsion was similar to CO samples analyzed by GC–MS (Table S Supporting Information). Donsì, Annunziata, Sessa, and Ferrari (2011) found a significant loss in the efficacy of antimicrobial compounds during processing of emulsion. Table 3 represents the MIC values against two GPB (L. monocytogenes and S. aureus) and GNB strains (E. coli) treated with CO and CO nanoemulsion using an agar dilution method. It was found that the MIC of PGU emulsified CO nanoemulsion against L. monocytogenes and S. aureus was 0.1% and 0.14% v/v, respectively. These MIC values were the same as that of CO. These results were in accordance with previous findings (Barbosa et al., 2009; Fu et al., 2007). However, a Tween 80-based CO nanoemulsion formulation showed greater MIC values of 0.12% and 0.16% v/v against L. monocytogenes & S. aureus compared to the CO and PGU CO nanoemulsion. These results suggest a decrease in the antimicrobial activity of CO when emulsified with a Tween 80 oil

Bacterial strain

b c

0.12

0.16

0.1

TSAYE: trypticase soy broth yeast extract. LB: Luria broth. PGU: purity gum ultra (modified starch).

in water emulsion system. On the other hand, PGU and Tween 80 emulsified CO nanoemulsions showed slight differences in MIC levels (0.1% and 0.08% v/v) against E. coli, a GNB strain. Conclusively, against E. coli both PGU and Tween 80 based nanoemulsion formulations showed compromised antimicrobial activity and a higher MIC value compared to CO. A time kill dynamic experiment was conducted to compare the bactericidal activities of either CO or CO nanoemulsions towards the growth of GPB (L. monocytogenes and S. aureus) and GNB strain (E. coli). The MIC of the CO and PGU emulsified CO nanoemulsion against GPB strains were same. However, in the case of the Tween 80 CO nanoemulsion, the MIC values were different compared to bulk CO. For GNB, the MIC value of CO was lower compared to the PGU and Tween 80 CO nanoemulsions as shown in Table 3.

80

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

The emulsifier solution showed no inhibitory effect on bacterial growth and showed the same level as in the growth control (data not shown). The addition of a CO or CO nanoemulsion strongly inhibited the growth of both GPB (L. monocytogenes & S. aureus) strains at the onset. In case of L. monocytogenes the bacterial number was reduced to approximately 101 CFU/ml during the first 8 h and later no significant bacterial growth was observed for 36 h. As shown in Fig. 3A, the bacterial growth was sluggish for the PGU emulsified CO nanoemulsion and the bacterial number decreased to almost 102 CFU/ml after 48 h. On the other hand, the bacterial number increased to 104 CFU/ml after 48 h with CO. However, when L. monocytogenes was treated with Tween 80 emulsified CO nanoemulsion, the bacterial number reduced to 103 CFU/ml after 10 h but, later no bacterial growth inhibition was observed and the bacterial number increased to 105 CFU/ml after 48 h (Fig. 3D). S. aureus showed less sensitivity to CO compared to L. monocytogenes (Fig. 3B). The bacterial number decreased to 101 CFU/ml after 10 h and this inhibitory effect continued until 24 h with CO nanoemulsion treatment. The bacterial number increased to 101 and 104 CFU/ml for CO nanoemulsion and CO treatments, respectively. S. aureus showed a similar trend to that of L. monocytogenes when treated with Tween 80 emulsified CO nanoemulsion. The bacterial number reduced to 103 CFU/ml after 12 h and then continuously increased and finally reached 104 CFU/ml after 48 h (Fig. 3E). Interestingly, both PGU and Tween 80 emulsified CO nanoemulsions remained ineffective against a GNB strain E. coli. For E. coli treated with PGU emulsified CO nanoemulsion and CO, depicted in Fig. 3C, the bacterial number decreased to 103 CFU/ml after 12 h and started to increase after CO nanoemulsion treatment. On the other hand, with CO treatment, the bacterial number decreased to 102 CFU/ml. Finally, the bacterial number increased to 104 CFU/ml after treatment with the PGU CO nanoemulsion, but lowered to 102 CFU/ml, with CO. On the other hand, when E. coli was treated with Tween 80 CO nanoemulsion the results showed compromised antimicrobial activity and CO was better at inhibiting the growth of E. coli compared to Tween 80 emulsified CO nanoemulsion (Fig. 3F). The results show that PGU CO nanoemulsions have the same MIC values against both GPB strains (L. monocytogenes and S. aureus) and inhibited their growth for a longer time. Moreover, the MIC values obtained against GNB (E. coli) and GPB strains (L. monocytogenes and S. aureus) were less for CO compared to Tween 80 CO nanoemulsion (Table 3). Time kill dynamic experiment results showed that CO was more effective at inhibiting the growth of GPB and GNB strains compared to Tween 80 CO nanoemulsion, as shown in Fig. 3D –F. This could probably be attributed to the different structure of the cell wall of GPB and GNB. GPB possesses a cell wall that mainly consists of teichoic acid, which contains more phosphate groups in its structure that confer polyanionic properties. For GNB, the outer membrane consists of lipopolysaccharides (LPS). Among these LPS, most phospholipids provide net negative charge to bacteria (Caroff & Karibian, 2003; Dunne, 2002). In a recent study interaction of positively charged polymeric materials with LPS of the GNB cell wall has been reported (Dillen et al., 2008). In our case, the negatively charged PGU CO nanoemulsion droplets might have interacted well with the LPS of the GPB cell wall and, therefore, showed better antibacterial activity. However, in case of GNB (E. coli) the LPS did not interact and showed compromised bactericidal action. A second reason may be a chemical linkage between teichoic acid of GPB with the octenyl succinylated anhydride group of PGU (structure shown in Fig. S3, Supporting Information). For E. coli, GNB lacks teichoic acid and therefore no chemical linkage was possible. On the other hand, Tween 80 is neutral and consequently would not interact with teichoic acid. In order to confirm this, we studied the adhesion of PGU and Tween 80 CO nanoemulsion droplets with GPB and GNB.

The adhesion of positively charged PLGA nanoparticles with the Pseudomonas aeruginosa and S. aureus has already been studied using this procedure. The size distributions of L. monocytogenes, S. aureus and E. coli in sterile PBS and after treatment with PGU and Tween 80 CO nanoemulsions are shown in Fig. S2 (Supplementary Information). The size and zeta potential in sterile PBS for L. monocytogenes (551 ± 4 & 17.4 mV), S. aureus (1108 ± 14 & 19 mV) and E. coli were (677 ± 7 & 22 mV). The average size of L. monocytogenes (470 ± 3) and S. aureus (989 ± 14) increased after treatment with negatively charged PGU CO nanoemulsion as shown in Fig. S2 (a and b). However, as can be seen from Fig. S2, the size distribution of E. coli with PGU CO nanoemulsion showed two separate peaks. For Tween 80 CO nanoemulsions size distribution showed two separate peaks of bacteria and nanoemulsion as shown in Fig. S2 (c, d & f). It has already been proved that when there is no interaction or slight adhesion between bacteria and nanoparticle two populations coexist during size measurement (Dillen et al., 2008). Kim, Gias, and Jones (1999) confirmed light scattering intensity of bacteria predominates over the nanoparticle when bacteria and nanoparticles were mixed and no interaction occurred. The size increase of S. aureus and L. monocytogenes after treatment with PGU CO nanoemulsion droplets pointed to a slight adhesion that suggests their interaction with bacterial cell wall components and negatively charged droplets. Since both bacteria (S. aureus and L. monocytogenes) and nanoemulsion droplets have negative surface charge, adhesion probably occurs through self-assembly or chemical linkage rather than electrostatic interaction. On the other hand, in the case of E. coli treated with PGU CO nanoemulsion, the results showed no adhesion and two separate peaks appeared which pointed to no adhesion. Moreover, all three bacteria (S. aureus, L. monocytogenes and E. coli) after treatment with Tween 80 CO nanoemulsion showed no interaction, which is probably due to no charge on droplets. However, the presence of two separate peaks for E. coli after treatment with negatively charged PGU CO nanoemulsion was surprising. These results suggest that PGU CO nanoemulsion droplets self-organize or chemically interact with the (S. aureus and L. monocytogenes) bacterial cells, disintegrate the cell wall and cytoplasmic membranes, and ultimately release cellular constituents. On the other hand, with E. coli PGU nanoemulsion droplets did not self-organize or chemically interact but, instead showed non-contact interaction. The same phenomenon of self-assembly between nanoparticles (gold & platinum) and bacteria (L. monocytogenes & Salmonella enteritidis) has already been reported (Chwalibog et al., 2010). Self-assembly takes place in various morphologic interactions, which depends on the type of nanoparticle and the microorganism used (Sawosz et al., 2010). To further confirm the bactericidal mechanism of both nanoemulsions these were visualized by fluorescence microscopy. Images of bacterial cells exposed to both PGU and Tween 80 CO nanoemulsions, loaded with fluorescent dye, were recorded after 24 h as shown in Fig. S3 (Supplementary Information). The nanoemulsions were difficult to visualize under fluorescent microscope due to their nanometric size, with only a fluorescent halo being observed. In contrast, when the nanoemulsion droplets accumulate in the cell membrane as well as in the intracellular space the bacterial cell (S. aureus & L. monocytogenes) became fluorescent as shown in Fig. S3 b and e. These results suggest that the PGU CO nanoemulsion interacts with the bacterial cell membrane. However, in the case of E. coli the cells remained non-fluorescent, which suggests noncontact interaction of PGU CO nanoemulsion (Fig. S3h). On the other hand, all three bacteria (S. aureus, L. monocytogenes and E. coli) after exposure with Tween 80 CO nanoemulsion remained viable and non-fluorescent. This suggests that the non-ionic small molecule surfactant adsorbed strongly on the droplet surface and restricted the leakage of essential oil components and, therefore, diminished the antimicrobial activity. The antimicrobial efficacy

81

10

8

10

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

10

-1

10

-2

D

A

log CFU/mL

log CFU/mL

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

0

10

20

30

40

50

10

8

10

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

0

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

10

-1

10

-2

log CFU/mL

10

0

10

9

10

8

10

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

10

20

30

40

50

0

10

8

10

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

0

T im e (H ours)

C

10

20

30

30

40

50

40

50

40

50

E

B

log CFU/mL

log CFU/mL

log CFU/mL

8

20

Time (Hours)

Time (Hours) 10

10

40

50

Time (Hours)

10

8

10

7

10

6

10

5

10

4

10

3

10

2

10

1

10

0

10

30

T im e (H ours)

F

0

20

10

20

30

Time (Hours)

Fig. 3. Time kill plots for both nanoemulsions against L. monocytogenes (A, D), S. aureus (B, E) and E. coli (C, F). The concentration of the oil phase in the nanoemulsions was 10% v/v, the ratio of CA to CO in the oil phase was 1:1, the surfactants were either PGU (A, B, C) or Tween 80 (D, E, F), and they were used at a 2% w/w concentration in the aqueous phase. The concentrations of the nanoemulsions used against bacterial strains were the minimum inhibitory concentrations (MIC). See Table 3.

of large and small molecule surfactant based oil in water emulsion has been confirmed previously by various studies (Donsi et al., 2012).

However, some researchers have reported contradictory results. Terjung et al. (2012) prepared phytophenols (eugenol and carvacrol) loaded nanoemulsions using a small molecule surfactant,

82

H. Majeed et al. / Food Chemistry 197 (2016) 75–83

Tween 80, and found that small nanoparticles (200 nm) were less effective than macroparticles (3000 nm). They suggested that sequestration of phytophenols at the interface resulted in less availability in the aqueous phase and ultimately limited their efficacy. In our case, small charged PGU-based nanoemulsion droplets were more effective and we hypothesize that it may be due to self-assembly or chemical interaction of negatively charged succinate anhydride-modified starch droplets with the microbial cell wall components. Further work is still being done to investigate the conformation of emulsifiers at the oil/water interface and to expose the morphologic interaction of negatively charged nanoparticles with bacterial cell membranes. In conclusion, stable negatively charged CO nanoemulsions were prepared using a CO and CA oil mixture. These nanoemulsions remained stable against coalescence and phase separation for an extended storage period. The MIC values obtained against GPB strains were similar for CO and CO:CA nanoemulsion. MIC values were also observed against GNB (E. coli). Our time kill studies showed enhanced antimicrobial activity of PGU-emulsified CO nanoemulsion against GPB (L. monocytogenes and S. aureus) strains compared to CO. These findings could lead to a more rational design of nanoemulsion based delivery systems for essential oils and the desired function of their constituents against food pathogens. Acknowledgements This work was financially supported by National 863 Program 2011BAD23B02, 2013AA102207, NSFC – China, 31171686, 30901000, 111 project-B07029 and PCSIRT0627. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 10.015. References Arana-sanchez, A., Espinosa, M. E., Obledo-vazquez, E. N., Padilla-Camberos, E., Silva-Vazques, R., & Lugo-Cervantes, E. (2010). Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated in b-cyclodextrin. Letters in Applied Microbiology, 50, 585–590. Babu, A. J., Sundari, A. R., Indunmathi, J., Srujan, R. V. N., & Sravanthi, M. (2011). Study on the antimicrobial activity and minimum inhibitory concentration of essential oils of spices. Veterinary World, 4(7), 311–316. Barbosa, L. N., Rall, V. L. M., Fernandes, A. A. H., Ushimaru, P. L., Probst, I. D., & Fernandes, A. J. (2009). Essential oils against foodborne pathogens and spoilage bacteria in minced meat. Foodborne Pathogens and Disease, 6(6), 725–728. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology, 94, 223–253. Burt, S. A., & Reinders, R. D. (2003). Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Letters in Applied Microbiology, 36, 162–167. Caroff, M., & Karibian, D. (2003). Structure of bacterial lipopolysaccharides. Carbohydrate Research, 338, 2431–2447. Chaieb, K., Hajlaoui, H., Zmantar, T., Kahla-Nakbi, A. B., Rouabhia, M., & Mahdouani, K. (2007). The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzygium aromaticum L. Myrtaceae): A short review. Phytotherapy Research, 2, 501–506. Chwalibog, A., Sawosz, E., Hotowy, A., Szeliga, J., Mitura, S., Mitura, K., et al. (2010). Visualization of interaction between inorganic nanoparticles and bacteria or fungi. International Journal of Nanomedicine, 5, 1085–1094. Dillen, K., Bridts, C., Veken, P. V. D., Cos, P., Vandervoort, J., Augustyns, K., et al. (2008). Adhesion of PLGA or EudragitÒ/PLGA nanoparticles to Staphylococcus and Pseudomonas. International Journal of Pharmaceutics, 349, 234–240. Donsì, F., Annunziata, M., Sessa, M., & Ferrari, G. (2011). Nanoencapsulation of essential oils to enhance their antimicrobial activity in foods. LWT – Food Science and Technology, 44, 1908–1914.

Donsi, F., Annunziata, M., Vincensi, M., & Ferrari, G. (2012). Design of nanoemulsion based delivery systems of natural antimicrobials: Effect of the emulsifier. Journal of Biotechnology, 159, 324–350. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: Antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88, 308–316. Dunne, W. M. (2002). Bacterial adhesion: Seen any good biofilms lately? Clinical Microbiology Reviews, 15, 155–166. Edris, A. E. (2007). Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: A review. Phytotherapy Research, 21, 308–323. Fu, Y., Zu, Y., Chen, L., Shi, X., Wang, Z., & Sun, S. (2007). Antimicrobial activity of clove and rosemary essential oils alone and in combination. Phytotherapy Research, 21, 989–994. Guarda, A., Rubilar, J. F., Miltz, J., & Galotto, M. J. (2011). The antimicrobial activity of microencapsulated thymol and carvacrol. International Journal of Food Microbiology, 146, 144–150. Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology, 86, 985–990. Hecht, L. L., Wagner, C., Landfester, K., & Schuchmann, H. P. (2011). Surfactant concentration regime in miniemulsion polymerization for the formation of MMA nanodroplets by high pressure homogenization. Langmuir, 27, 2279–2285. Hill, L. E., Gomes, C., & Taylor, T. M. (2013). Characterization of beta-cyclodextrin inclusion complexes containing essential oils (trans-cinnamaldehyde, eugenol, cinnamon bark, and clove bud extracts) for antimicrobial delivery applications. LWT – Food Science and Technology, 51, 86–93. Jones, S. F. A. (1996). Herbs Useful plants. Their role in history and today. European Journal of Gastroenterology and Hepatology, 8, 1227–1231. Joseph, B., & Sujatha, S. (2011). Bioactive compounds and its autochthonous microbial activities of extract and clove oil (Syzygium aromaticum L.) on some food borne pathogens. Asian Journal of Biological Sciences, 4(1), 35–43. Kabalnov, A. (2001). Ostwald ripening and related phenomena. Journal of Dispersion Science Technology, 22, 1–12. Kim, H. J., Gias, E. L. M., & Jones, M. N. (1999). The adsorption of cationic liposomes to Staphylococcus aureus biofilms. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 149, 561–570. Liang, R., Xu, S., Shoemaker, C. F., Li, Y., Zhong, F., & Huang, Q. (2012). Physical and antimicrobial properties of peppermint oil nanoemulsions. Journal of Agriculture and Food Chemistry, 60, 7548–7555. LisBalchin, M., & Deans, S. G. (1997). Bioactivity of selected plant essential oils against Listeria monocytogenes. Journal of Applied Microbiology, 82, 759–762. Mao, L., Xu, D., Yang, J., Yuan, F., Gao, Y., & Zhao, J. (2009). Effects of small and large molecule emulsifiers on the characteristics of b-carotene nanoemulsions prepared by high pressure homogenization. Food Technology and Biotechnology, 47, 336–342. McClements, D. J., & Rao, J. (2011). Food-grade nanoemulsions: Formulation, fabrication, properties, performance, biological fate, and potential toxicity. Critical Reviews in Food Science and Nutrition, 51, 285–330. Mekkerdchoo, O., Patipasena, P., & Borompichaichartkul, C. (2009). Liposome encapsulation of antimicrobial extracts in pectin film for inhibition of food spoilage microorganisms. Asian Journal of Food Agriculture Industry, 2(4), 817–838. Moon, S., Kim, H., & Cha, J. (2011). Synergistic effect between clove oil and its major compounds and antibiotics against oral bacteria. Archives of Oral Biology, 56, 907–916. Mytle, N., Anderson, G. L., Doyle, M. P., & Smith, M. A. (2006). Antimicrobial activity of clove (Syzygium aromaticum) oil in inhibiting Listeria monocytogenes on chicken frankfurters. Food Control, 17, 102–107. Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2011a). Comparison of biopolymer emulsifier performance in formation and stabilization of orange oil in water emulsions. Journal of American Oil Chemical Society, 88, 47–55. Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2011b). Physical and chemical stability of b-carotene-enriched nanoemulsions: Influence of pH, ionic strength, temperature, and emulsifier type. Food Chemistry, 132, 1221–1229. Rodriguez-Rojo, S., Varona, S., Nunez, M., & Cocero, M. J. (2012). Characterization of rosemary essential oil for biodegradable emulsions. Industrial Crops and Products, 37, 137–140. Ru, Q., Yu, H., & Huang, Q. (2010). Encapsulation of epigallocatechin-3-gallate (EGCG) using oil-in-water (O/W) submicrometer emulsions stabilized by icarrageenan and b-lactoglobulin. Journal of Agriculture and Food Chemistry, 58, 10373–10381. Sawosz, E., Chwalibog, A., Szeliga, J., Sawosz, F., Grodzik, M., Rupiewicz, M., et al. (2010). Visualization of gold and platinum nanoparticles interacting with Salmonella enteritidis and Listeria monocytogenes. International Journal of Nanomedicine, 5, 631–637. Shah, B., Davidson, P. M., & Zhong, Q. (2013). Nanodispersed eugenol has improved antimicrobial activity against Escherichia coli O157:H7 and Listeria monocytogenes in bovine milk. International Journal of Food Microbiology, 161, 53–59. Solomon, B., Sahle, F. F., Gebre-Mariam, T., Ares, K., & Neubert, R. H. H. (2012). Microencapsulation of citronella oil for mosquito-repellent application: Formulation and in vitro permeation studies. European Journal of Pharmaceutics and Biopharmaceutics, 80, 61–66.

H. Majeed et al. / Food Chemistry 197 (2016) 75–83 Tan, C., & Nakajima, M. (2005). B-carotene nanodispersions: Preparation, characterization and stability evaluation. Food Chemistry, 92, 661–671. Terjung, N., Loeffler, M., Gibis, M., Hinrichs, J., & Weiss, J. (2012). Influence of droplet size on the efficacy of oil-in-water emulsions loaded with phenolic antimicrobials. Food Function, 3, 290–301.

83

Ultee, A., Bennik, M. H. J., & Moezelaar, R. (2002). The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Applied and Environmental Microbiology, 68, 1561–1568. Yuan, Y., Gao, Y., Zhao, J., & Mao, L. (2008). Characterization and stability evaluation of b-carotene nanoemulsions prepared by high pressure homogenization under various emulsifying conditions. Food Research International, 41, 61–68.