Long-term stability of food-grade nanoemulsions from high methoxyl pectin containing essential oils

Food Hydrocolloids 52 (2016) 438e446

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Long-term stability of food-grade nanoemulsions from high methoxyl pectin containing essential oils
s Guerra-Rosas a, Juliana Morales-Castro a, Luz Araceli Ochoa-Martínez a, María Ine Laura Salvia-Trujillo b, Olga Martín-Belloso b, * a b


gico de Durango, Blvd. Felipe Pescador 1830, Ote., 34080 Durango, Mexico Departamento de Ingenierías Química y Bioquímica, Instituto Tecnolo Department of Food Technology, Universidad de Lleida e Agrotecnio Center, Av. Alcalde Rovira Roure 191, 25198 Lleida, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 February 2015 Received in revised form 12 June 2015 Accepted 16 July 2015 Available online 26 July 2015

Nanoemulsions have shown potential advantages over conventional emulsions due to their large active surface area, but are also susceptible to destabilization. Therefore, the purpose of this work was to assess the long-term stability (56 days) of nanoemulsions containing EOs (oregano, thyme, lemongrass or mandarin) stabilized by high methoxyl pectin and a non-ionic surfactant (Tween 80). The initial droplet size of nanoemulsion was below 50 nm regardless the EO type, which was confirmed by Transmission Electron Microscopy (TEM). Lemongrass and mandarin nanoemulsions remained optically transparent over time (56 days) and their droplet sizes were in the nano-range (between 11 and 18 nm), whereas the droplet size of oregano and thyme nanoemulsions increased up to 1000 nm probably due to Ostwald ripening. This fact induced creaming and a higher whiteness index in the latter nanoemulsions. The electrical charge (æ-potential) of nanoemulsions was negative due to the anionic nature of pectin molecule adsorbed at the oil-water interface, ranging between
6 and
15 mV depending on the EO type. However, lemongrass and mandarin nanoemulsions exhibited a more negative æ-potential than thyme or oregano EO indicating a stronger adsorption of pectin at the oil surface, and therefore a higher stability. The viscosity of nanoemulsions remained practically constant between 20 and 24 mPa s, during storage for all EOs. This work represents the starting point for future applications of nanoemulsions containing EOs to be incorporated in food products due to their high long-term stability. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Nanoemulsions Essential oils Droplet size z-potential Viscosity Creaming index

1. Introduction Essential oils (EOs) are natural compounds found in aromatic plants and herbs as secondary metabolites that present antioxidant and antimicrobial activity and also have been widely used as functional ingredients in food as flavorings (Burt, 2004). However their incorporation in food products presents several limitations due to their low solubility and intense aroma at high concentrations
nchez-Gonza
lez, Vargas, Gonza
lez-Martínez, Chiralt, & Cha
fer, (Sa 2011). The emulsification of EO is currently used for their dispersion into food products but their functionality and long-term stability largely depends on the oil droplet size and distribution (Tadros, Izquierdo, Esquena, & Solans, 2004). In this sense, nanoemulsions can be used as carriers of lipophilic bioactive compounds for their incorporation in food products. Nanoemulsions consist of

* Corresponding author. E-mail address: [email protected] (O. Martín-Belloso). http://dx.doi.org/10.1016/j.foodhyd.2015.07.017 0268-005X/© 2015 Elsevier Ltd. All rights reserved.

at least one immiscible liquid dispersed in another with a surfactant (nonionic or polymeric) in the form of small droplets, with an average droplet size between 20 and 200 nm (Burguera & Burguera, 2012; Solans, Izquierdo, Nolla, Azemar, & Garcia-Celma, 2005;
rez, Torcello-Go
mez, G
Wulff-Pe alvez-Ruíz, & Martín-Rodríguez, 2009). Nanoemulsions exhibit several advantages over conventional emulsions (Qian & McClements, 2011; Tadros, Izquierdo, Esquena, & Solans, 2004). First, they are optically transparent so they might be good candidates to be incorporated in clear drinks or beverages (Qian & McClements, 2011). Second, nanoemulsions are kinetically stable colloidal systems (Solans et al., 2005). Third, they present a high active surface area thus having a potentially higher functionality (Qian & McClements, 2011). There are several methods to form nanoemulsions, but high-energy methods are the most commonly used. They require specialized mechanical devices such as high-pressure homogenizers and ultrasounds capable of generating intense mechanical disruptive forces inducing the breakup of the oil droplets (Mason, Wilking, Meleson, Chang, &

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

Graves, 2006; Tadros et al., 2004). Nevertheless, nanoemulsions are susceptible to destabilization phenomena, so an optimal formulation is crucial for their long term stability (Henry, Fryer, Frith, & Norton, 2009; Mirhosseini et al., 2008). Stability may be defined as the resistance to physical changes. It has been reported that emulsions are destabilized via two mechanisms, being coalescence and Ostwald ripening (Henry et al., 2009; Nazarzadeh, Anthonypillai, & Sajjadi, 2013; Solans et al., 2005). Coalescence occurs when two oil droplets contact due to the weak stearic repulsion between them, and they unify in a sole larger droplet. Ostwald ripening is due to the diffusion of oil molecules from small to large droplets through the continuous phase in relatively watersoluble oils (such as EOs), leading to an increase in the oil droplet size (Nazarzadeh et al., 2013; Rao & McClements, 2012). On the other hand, food hydrocolloids such as polysaccharides and proteins have been used to stabilize emulsions in several studies (Ye & Singh, 2006). Pectin is a naturally-sourced polysaccharide and is commonly used in the food and pharmaceutical industries as gelling and thickening agent (Liu, Fishman, & Hicks, 2007). In addition, high methoxyl pectin can be used as emulsi
rez-Espitia, fying agent (Liu et al., 2007; Mirhosseini et al., 2008; Pe Du, Avena-Bustillos, Ferreira Soares, & McHugh, 2014; Sungthongjeen, Sriamornsak, Pitaksuteepong, Somsiri, & Puttipipatkhachorn, 2004). Pectins have amphiphilic character that helps to reduce the interfacial tension between oil and water phases and can be effective and suitable in the preparation and formulation of emulsions (Burapapadh, Kumpugdee-Vollrath, Chantasart, & Sriamornsak, 2010). Therefore, the aim of the present work was to study the stability of food-grade nanoemulsions containing essential oils (oregano, thyme, mandarin and lemongrass) to determine what type of nanoemulsions remained without changes during the storage time. For this purpose, we evaluated the physicochemical characteristics and overall long-term stability (56 days at room temperature) of the nanoemulsions. 2. Material and methods 2.1. Materials Essential oils from oregano (Origanum compactum) and thyme (Thymus vulgare) were purchased from Dietetica Intersa (Spain), whereas lemongrass (Cymbopogon citratus) was obtained from Laboratories Dicana (Spain). Mandarin EO (Citrus reticulata) was kindly donated by Indulleida, S.A. (Spain). Food-grade high methoxyl pectin (Unipectine QC100 from citrus source) was provided by Cargill Inc. (Spain). Tween 80 (Polyoxyethylenesorbitan Monoesterate) (Lab Scharlab, Spain) was used as food-grade nonionic surfactant. Ultrapure water, obtained from Millipore Milli-Q filtration system (0.22ìm) was used for the formulation and analysis of nanoemulsions. 2.2. Primary emulsion formation The independent variables used were: emulsion formulation and oregano, thyme, lemongrass and mandarin essential oils. High methoxyl pectin (1% w/v) was dissolved in water at 80e85  C, with continuous stirring until it was completely dissolved and the solution was cooled down to 25  C. A primary emulsions was made mixing the pectin aqueous solution and the essential oil (2% v/v) and Tween 80 (5% w/v) by means of a laboratory T-25 digital Ultraturrax (IKA, Staufen, Germany) working at 9500 rpm for 2 min. The final volume of the primary emulsion was 1000 mL. Droplet size and droplet size distribution, Z-potential, creaming index, color, viscosity and transmission electron microscopy were measured and were considered as dependent variables (Table 1).

439

2.3. Nanoemulsion formation Nanoemulsions were obtained by microfluidization (M-110P, Microfluidics, USA) at 150 MPa for 5 cycles. Nanoemulsions were cooled down at the outlet of the microfluidization unit through an external coil immersed in a water bath with ice, so that temperature was kept at 10  C. The final volume of each nanoemulsion was 950 mL, as 50 mL were discarded to avoid the dilution of the sample. For stability studies, aliquots of nanoemulsions were placed in plastic test tubes with caps and stored at room temperature (~25 ± 2  C) in the absence of light. Analytical determinations were performed right after preparation and along storage time (0, 7, 14, 21, 28, 35, 42, 49 and 56 days). When creaming occurred, nanoemulsions were homogenized to re-disperse the cream layer before the analysis. 2.4. Nanoemulsion characterization 2.4.1. Droplet size and droplet size distribution The average droplet size of the nanoemulsion was determined by dynamic-light-scattering (DLS), using a Zetasizer Nano-ZS laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK), working at 633 nm, equipped with a backscatter detector (173 ), which is used to specifically measure sub-micron particles. The DLS measures particle diffusion moving under Brownian-motion. Nanoemulsions were diluted 100 times with milli-Q water to avoid multiple-scattering effect and stirred to ensure sample homogeneity. DTS0012-disposable cuvettes were used, which the minimum volume sample is required (1 mL). The refractive indexes (RI) of the oil phases were measured with a manual refractometer (model J357, Rudolph research, New Jersey, USA) being 1.501, 1.497, 1.484 and 1.475 for the oregano, thyme, lemongrass and mandarin EOs, respectively. The absorbance of the EOs at 633 nm was measured with a spectrophotometer Cecil CE 1021 (Cambridge, England) being 0.002, 0.002, 0.024 and 0.004 for the oregano, thyme, lemongrass and mandarin EOs, respectively. Droplet-size measurements are reported as average-volume. Polydispersity Index (PdI) was also recorded, a value near 1 indicates a heterogeneous or multimodal distribution of droplet sizes. Determinations were performed at 25  C, 24 h after the preparation of the nanoemulsions and every 7 days up to 56 days. 2.4.2. Transmission electron microscopy Nanoemulsions were observed by negative-staining electron microscopy as a direct measure of their droplet size and shape. The sample was adsorbed onto carbon film on 300 mesh copper grids for 1 min. Then, the grid was washed by floating it face-down on a drop of Milli-Q ultrapure water for 1 min. This process was repeated three times. Finally, the sample was negatively stained by floating the grid face-down on a drop of 2% (w/v) ammonium molybdate at pH 6.2 for 1 min. The images of the samples were obtained observing the grids in a Jeol-JEM 1010 transmission electron microscope (Biodirect, Inc., Massachusetts, USA) at an acceleration voltage of 100 kV. 2.4.3. Particle charge measurements (z-potential) The electrical charge (æ-potential) of the oil droplets in the nanoemulsions was determined by phase-analysis light scattering (PALS) measuring their electrophoretic mobility using an automated capillary electrophoresis device (Zetasizer Nano ZS series, Malvern Instruments Ltd, Worcestershire, UK) and working at 633 nm laser at 25  C. Sample was placed in a disposable zeta cuvette that acted as a measurement chamber, and the æ-potential was determined by measuring the particle's direction and velocity when an electric field was applied. The Smoluchowski model was

440

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446 Table 1 Independent and dependent variables. Independent variables

Dependent variables

 Nanoemulsion formulation  Oregano, thyme, lemongrass and mandarin essential oils

     

used by the instrument's software program to convert the electrophoretic mobility measurements into æ-potential values. The measurements were performed after the preparation of the nanoemulsion.

Droplet size and droplet size distribution Z-potential Creaming index Color Viscosity Transmission electron microscopy

(0, 7, 14, 21, 28, 35, 42, 49 and 56 days), at 5% significant level (interval of confidence of 95%). All results were expressed as mean ± standard deviation. 3. Results and discussion

2.4.4. Creaming index Creaming index (%) for each nanoemulsion during time was determined as described by Mirhosseini et al., (2008), with some modifications (Equation (1)). Each nanoemulsion (15 mL) was filled into a glass tube sealed with a cap (height 60 mm and 22 mm internal diameter) and stored at ambient temperature (~25 ± 2  C) in quiescent conditions. The height of cream layer (Hc) and total height of nanoemulsion (He) were measured with a Mitutoyo vernier pattern (Japan) during storage time. The results were expressed as the percentage of creaming index (CI) using the Equation (1):

 CI ¼

 Hc  100 He

(1)

2.4.5. Color The color of nanoemulsions was assessed at room temperature (~25  C), using a Minolta Chroma Meter CR-400 colorimeter (Konica Minolta Sensing, Inc., Osaka, Japan) with an illumination D65 and 10 observer angle, to determine the L* value (lightness), a* value (redness) and b* value (yellowness). A white standard (tile: L* ¼ 93.24, a* ¼
0.72, and b* ¼ 1.53) was used to calibrate the colorimeter before the measurement was taken. The results were expressed as mean values of whiteness index (WI), calculated with
fer, Albors, Chiralt, & Gonza
lez-Martínez, Equation (2) (Vargas, Cha 2008):

0:5   WI ¼ 100
ð100
LÞ2 þ a2 þ b2

(2)

2.4.6. Viscosity Viscosity of nanoemulsions was measured of an aliquot of 10 mL of sample using an SV-10 Vibro-Viscometer (A&D Company, Tokyo, Japan), working with a vibration at 30 Hz and constant amplitude. Water viscosity (used as dispersant phase), being 0.8872 mPa s, was considered with regard to DLS measurements. Measurements were performed at 25 ± 2  C. 2.4.7. Statistical analysis All analysis were done in duplicate, and three replicate analyses were carried out on each parameter at 25 ± 2  C in order to obtain mean values. Statistical analysis of experimental data was done by a multifactor analysis of variance using Statgraphics Plus software version 5.1, Windows package (Statistical Graphics Co., Rockville, MD) and was compared using the least significant difference (LSD) test to determine differences among EOs and along the storage time

3.1. Particle size and particle size distribution In the present study the average droplet size and its distribution of the nanoemulsions and primary emulsions were assessed during storage time. Significant differences were observed on the droplet size of primary emulsions and nanoemulsions depending on the EO type. The average droplet size of the primary emulsions containing oregano, thyme, lemongrass or mandarin EO was 740.40 ± 268.10, 82.09 ± 23.62, 370.76 ± 16.72 and 601.46 ± 7.89 nm, respectively. After passing the primary emulsions through the microfluidizer, the droplet size was significantly reduced down to 27.53 ± 12.59, 40.60 ± 8.71, 11.89 ± 0.84 and 17.56 ± 1.22 nm, respectively. Microfluidization process reduces the average droplet size, size distribution and PdI (Jafari, He, & Bhandari, 2006; Salvia-Trujillo, Rojas-Graü, Soliva-Fortuny, & Martín-Belloso, 2013;). Regarding the stability of the nanoemulsions, in Table 2, it can be seen that the data with the same superscript within the same row does not show significant differences over time. In the case of the pectin nanoemulsion containing lemongrass EO, it showed no change in the values of this parameter during the storage period. The same behavior was observed in the pectin nanoemulsion containing mandarin EO, which at the beginning of the study period (day zero) presented a value of 17.56 ± 1.22 nm, changing its value to 13.61 ± 1.34 nm (day 7). This last nanoemulsion only showed a significant difference at baseline and remained practically unchanged for the rest of the storage period. Both nanoemulsions showed the same behavior during the 56 days of storage at room temperature indicating a high stability. Klang, Matsko, Valenta, and Hofer (2012) reported that, when droplet size and its distribution remain practically constant during an extended observation period, a formulation is usually considered physically stable. By contrast, droplet size values of nanoemulsions containing oregano or thyme EOs increased after seven days of storage up to 169.30 ± 10.69 and 105.10 ± 45.43 nm, respectively and, by the end of storage, it increased significantly up to 1017.00 ± 198.40 and 924.10 ± 183.80 nm, respectively. The significant droplet size increase observed in thyme or oregano EO nanoemulsions led to the separation of a serum and a cream layer over time (Fig. 2). This fact might be probably due to the ability of oil molecules to migrate from smaller droplets to larger droplets through the aqueous phase, known as Ostwald ripening effect (Taylor, 1998). EOs containing carvacrol in its composition are specifically prone to Ostwald ripening due to its relatively high solubility in water. In fact, Ziani, Chang, Mclandsborough, and McClements (2011) found that thyme oil-in-water nanoemulsions stabilized by a non-ionic surfactant were highly unstable to droplet growth and phase separation and they attributed this phenomenon to Ostwald ripening. Furthermore, this phenomenon could be due to differences between

1017.00 ± 198.40d 924.10 ± 183.80d 12.31 ± 0.71abc 13.17 ± 1.72abc 188.30 265.20de 0.39ab 0.57a 1083.00 1044.00 11.87 12.35

± ± ± ±

d

49

865.50 1341.00 12.45 12.16

± ± ± ±

235.50 768.40f 1.71ab 0.79a

c

42

± ± ± ± 593.70 902.10 12.70 12.72 24.37 5.33c 0.88ab 1.19bc

± ± ± ± 225.00 117.00 12.09 13.57 20.37 5.67b 0.39bc 0.68c

± ± ± ± 226.30 106.70 12.36 14.25 10.69 45.43b 0.50a 1.34bc

± ± ± ± 169.30 105.10 11.33 13.61 12.59 8.71a 0.84ab 1.22d

± ± ± ± 27.53 40.60 11.89 17.56 Oregano Thyme Lemongrass Mandarin

Data shown are a mean ± standard deviation. Values in a row with the same superscript are not significantly different (P < 0.05).

105.60 131.70d 1.47c 0.71ab

952.60 1123.00 11.54 12.16

± ± ± ±

353.10 255.30e 0.33ab 0.56a

d

35

c

28

b

21

b

14

b

7

a

Storage time (days)

0

The creaming index (CI) gives indirect information about the extent of droplet aggregation, coalescence and flocculation in emulsions (Seta, Baldino, Gabriele, Lupi, & de Cindio, 2013; Ye & Singh, 2006). CI phenomenon is represented by the separation of oil droplets where a layer is placed on the top of the emulsion and is attributed to the difference in density between the dispersed and continuous phases by action of gravity force (Dickinson, Golding, & Povey, 1996; Robins, 2000). In our study, creaming was expressed as percentage of creaming index (% CI) and the results are presented in Fig. 3. As expected, the nanoemulsions showed different behavior to creaming during the storage time depending on the oil type used. Nanoemulsions of pectin containing lemongrass or mandarin EOs did not exhibit creaming during 56 days of storage time, presenting a high-stability due to their small droplet size. Nano-sized droplets present a significant reduction in the gravity force and the Brownian motion that may be sufficient for overcoming gravity (Sagalowicz & Leser, 2010; Tadros et al., 2004). On the other hand, nanoemulsions of pectin containing oregano and thyme EO were highly unstable and both presented a similar behavior. After 7 days of storage the CI of nanoemulsions containing oregano or thyme EO were 17.08 ± 1.01 and 6.50 ± 0.40%, respectively, presenting a cream layer on the top. Typically the migration of emulsion droplets to the top of the sample happens in few hours after emulsification (Tadros et al., 2004). The CI in emulsions with oregano or thyme EOs decreased over storage time down to values of 8.48 ± 0.24 and 7.43 ± 0.38%, respectively, probably due to that oil droplets become more compact over time. In this case we observed three layers in the test tubes, being a cream layer on the top, a serum phase (with a low concentration of oil droplets) and a sediment of particles at the bottom (Fig. 1). The fast creaming

Essential oil

3.2. Creaming index

Table 2 Changes in the average droplet size (nm) values of essential oil-pectin nanoemulsions during storage time at 25  C.

density of continuous and dispersed phase (Dickinson & Ritzoulis, 2000; Robins, 2000). The droplet size reduction after microfluidization of primary emulsions was also confirmed with the PdI values (Fig. 2), where those nanoemulsions that presented smaller droplet sizes also showed smaller PdI. As it has been reported, the stability of nanoemulsions is strongly associated with their droplet size and PdI, considering stable emulsions or nanoemulsions when they remain unchanged over time (Klang et al., 2012). PdI values below 0.2 indicate uniformity among oil droplet sizes or monomodal distributions and therefore better stability whereas values close to 1 indicate a heterogeneous or multimodal distribution (Hoeller, Sperger, & Valenta, 2009; Klang et al., 2012). In this study, PdI values for the primary emulsions with oregano, thyme, lemongrass and mandarin EOs were 0.38 ± 0.04, 0.53 ± 0.03, 0.62 ± 0.12 and 0.70 ± 0.12, respectively. These values changed after microfluidization, producing particles that tend to stabilize or agglomerate depending on EOs used. In the case of emulsions containing lemongrass and mandarin EOs, PdI decreased down to 0.61 ± 0.15 and 0.57 ± 0.09, respectively; these values obtained indicate more uniform sizes, being corroborated by the reduced droplet size in these nanoemulsions. On the contrary, for nanoemulsions containing oregano and thyme, PdI values increased slightly up to 0.41 ± 0.02 and 0.63 ± 0.03, respectively, where the PdI changes throughout time implies a wider distribution due to increased particle size through the storage time, therefore there are particles that are out of range of ZetaSizer Nano-ZS laser diffractometer. In addition, a nanoemulsion may be polydispersive due to large difference in refractive index between dispersed and continuous. Overall, the decrease of particle size and PdI can improve or impair properties such as particle stability, viscosity, color, creaming and appearance of the nanoemulsions (Qian & McClements, 2011).

441

56

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

442

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

same time, thus getting them together, and causing flocculation
s, Vargas, & and consequently creaming of emulsions (Bonilla, Atare Chiralt, 2012). Robins (2000) reviewed the mechanism of creaming and phase separation due to its commercial importance in food emulsions. The absolute rates and degree of creaming and serum separation depend on the overall oil volume fraction of the nanoemulsion, its droplet-size distribution, and the detailed nature of the inter droplet interactions, including any effects of unabsorbed polymers and surfactants. Some authors have reported that the long-term stability of the colloidal dispersions formed depend also on the hydrocolloid used, such as the studies reported by Mirhosseini et al., (2008), where they evaluated the influence of pectin and carboxymethylcellulose (CMC) on physical stability of an orange beverage emulsion stored during two months at 25  C, they observed that the pectin was more effective on physical stability than CMC, due to the positive effect of pectin on æ-potential (repulsive forces). They concluded that pectin can enhance the electrostatic repulsive forces between emulsion droplets because it is a polysaccharide negatively charged. 3.3. Transmission electron microscopy (TEM)

1.00 Oregano

Thyme

Lemongrass

Mandarin

0.80

PdI

0.60 0.40 0.20 0.00 0

7

14

21

28

35

42

49

56

63

Storage time (days) Fig. 2. Polydispersity index (PdI) values of essential oil-pectin nanoemulsions during storage time at 25  C. Data shown are a mean ± standard deviation.

observed in nanoemulsions with oregano or thyme EOs might be related to the low electrostatic repulsion between oil droplets due to the weak charge at the oil-water interface (see discussion in the next section of æ-values). Oppositely, nanoemulsions with lemongrass and mandarin EOs presented a higher interfacial charge, thus leading to a stronger electrostatic repulsion between droplets and as a consequence a higher stability against creaming. The electrical charge of oil droplets is given by the adsorption of species at the oil-water interface. It is known that pectin can adsorb at the oil-water interface depending on its affinity with each oil type and give negative charge to the oil droplets. However, it is reported that when it is not absorbed, the presence of high concentrations of pectin in the aqueous phase leads to bridging flocculation phenomena, which is characteristic of many biopolymers (Mirhosseini et al., 2008; Robins, 2000). The chain-like molecular structure of biopolymers is able to adsorb to two oil droplets at the

20 16

12

CI (%)

Fig. 1. Appearance of the essential oil-pectin nanoemulsions during storage time at 25  C. (a) Oregano, (b) Thyme, (c) Lemongrass and (d) Mandarin.

Droplet size of nanoemulsions is typically measured by DLS, which is an indirect measurement considering the Brownian motion of lipid droplets and the light scattering. Therefore, microscopy techniques are often useful to characterize nanoemulsions, as a complementary tool in order to have a direct observation of the lipid particles and obtain reliable data about the morphology of the system (Klang et al., 2012). In this study, TEM was used to confirm the droplet size measured by DLS. Fig. 4 shows TEM images of fresh EO-pectin nanoemulsions produced by microfluidization (150 MPa and 5 cycles), where can be observed that the droplets, with spherical shape, in the nanoemulsions appear bright in a dark background. From the images obtained, it is relevant to point out that most of the droplets have a diameter below 50 nm (as can be seen in Fig. 5), which is in agreement with the particle size distribution from DLS measurement (Table 2), observing droplets with a size range around 100e150 nm with just a few big droplets. Accordingly to our results, Salvia-Trujillo et al. (2013) reported droplet sizes of 7.35 nm (determined by DLS) for nanoemulsions processed at 150 MPa and 3 cycles or passes, whereas the same nanoemulsions observed by TEM or AFM (atomic force microscopy) presented values between 10 nm and 250 nm of diameter. In a €der study conducted by Preetz, Hauser, Hause, Kramer, and Ma (2010) about the application of microscopy techniques on nanoemulsions and nanocapsules, they reported that the mean droplet size (by DLS) was approximately 150 nm for all studied systems and

8 4 0 Oregano

Thyme

Lemongrass

Mandarin

-4 0

7

14

21

28

35

42

49

56

63

Storage time (days) Fig. 3. Changes in creaming index (CI) values of essential oil-pectin nanoemulsions (produced by microfluidization at 150 MPa and 5 cycles) during storage time at 25  C. Data shown are a mean ± standard deviation.

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

when these were observed through freeze-fracture TEM, droplet sizes were between 50 and 500 nm observing great variations, which were additionally confirmed by atomic force microscopy. Thus, they demonstrated the importance of microscopic analysis for the characterization of nanoemulsions and nanocapsules. Therefore, obtaining nanoemulsions with a uniform droplet size becomes essential to achieve a stable system. 3.4. Particle charge measurements (z-potential) Zeta potential (æ-potential) is the difference in the electrical charge between the dense layer of ions surrounding the particles and the charge of the bulk of the suspended fluid surrounding this particle (Lu et al., 2005). The æ-potential is a measure of the repulsive forces between particles, and since the majority of colloidal aqueous systems are stabilized via electrostatic repulsion, the larger the repulsive forces between particles the lower the probability for them to become closer and form aggregates, leading to a more stable colloidal system (Heurtault, Saulnier, Pech, Proust, & Benoit, 2003). In theory, if the absolute value of æ-potential of particles is below 30 mV, a nanoemulsion exhibits a weak stability (agglomeration-flocculation), whereas for absolute values higher than 30 mV nanoemulsions are assumed to be stable due to electrostatic repulsion (Vallar, Houivet, Fallah, Kervadec, & Haussonne, 1999). In this study, the initial interfacial electrical charge values for the primary emulsions were
5.85 ± 0.52,
7.72 ± 0.32,
8.88 ± 1.17 and
9.11 ± 1.72 mV with oregano, thyme, lemongrass or mandarin EO-pectin, respectively. However, after microfluidization nanoemulsions with lemongrass or mandarin EOs presented a æ-potential of
15.20 ± 1.60 and
14.40 ± 0.57 mV, whereas oregano and thyme EOs showed a much weaker æ-potential with values of
6.53 ± 0.60 and
10.70 ± 1.17 (Fig. 6). æpotential differences between EOs may be due to different adsorption of pectin molecules at the interface, where the presence of pectin can enhance the electrostatic repulsive forces between emulsion droplets. The initial æ-potential of nanoemulsions of pectin with lemongrass or mandarin after microfluidization remained practically constant during storage time, whereas those with oregano or thyme decreased slightly over time. In fact, despite that the electrical charge of nanoemulsions with lemongrass or mandarin was not below
30 mV, the small droplet size presented was sufficient to prevent instability phenomena over storage time. Oppositely, nanoemulsions with thyme or oregano EOs presented æ-potential values of
2.68 ± 0.27 and
7.30 ± 0.40 mV respectively after 56 days. The lower electric charge presented by oregano or thyme nanoemulsions might be related with the creaming observed. The greater the absolute value of æ-potential the more likely the nanoemulsion is to be stable because the charged particles repel each other and thus overcome the natural tendency to aggregate. The æ-potential allows predictions to be made about the storage stability of a colloidal dispersion. For particles, an electrostatic repulsion occurs when the diffuse layers overlap while attraction is due to Van der Waals energy (Heurtault et al., 2003). The nature of surfactants controls the droplet charge (Yao et al., 2013); non-ionic surfactants like Tween 80 are usually used to stabilize the emulsions. The surfactants tend to deposit onto the oil-water interface due to their amphiphilic structure and reduce interfacial tension, facilitating droplet disruption, and protecting droplets against aggregation (Ktalova & €blom, 2009). Although the non-ionic surfactants do not provide Sjo electrical charge to oil droplet surface (Hsu & Nacu, 2003), some studies suggest that they can favor negative electrical charges, which can be attributed to the presence of anionic impurities (such as free fatty acids) in the oil or surfactant ingredients (Mayer, Weiss, & McClements, 2013). In this study, nanoemulsions containing

443

lemongrass and mandarin had æ-potential values around
15 mV, due to pectin adsorption. While in nanoemulsions with oregano or thyme EOs, the droplets had a relatively small negative charge and there is not pectin adsorption, therefore flocculation is favored. Same behavior has been reported recently by other authors studying the formation of nanoemulsions stabilized with Tween 80, where oil droplets coated by non-ionic surfactants have a slight negative charge (Mayer et al., 2013). Nevertheless, in the present work, the relatively low æ-potential values for oregano and thyme EOs nanoemulsions were not sufficient to achieve electrostatic stabilization and therefore, being highly unstable, presenting particle aggregation and sedimentation. Tween 80, in spite of being an anionic surfactant can give negative charge to the nanoemulsions (Hsu & Nacu, 2003), but usually close to
5 mV, at most. So the values close to
15 mV for nanoemulsions with mandarin and lemongrass can be due to the adsorption of pectin molecules at the interface, while in the case of thyme there is less adsorption, and in the case of oregano, practically there is not adsorption. The pectin molecule have carboxyl groups that at intermediate pH ~4.5 can lose protons and remain negatively charged, leading to a decrease in æ-potential (Chen et al., 2012). 3.5. Whiteness index Emulsions are widely used in food industry as controlled delivery systems in aqueous-based foods and beverages. One characteristic that is highly desirable is transparency while at the same time, stability must be maintained; therefore, nanoemulsions should be evaluated in regard to color parameters. In our study, color of nanoemulsions was measured and it was expressed as whiteness index (WI). The WI of fresh primary emulsions, was 55.79 ± 0.12, 43.29 ± 0.06, 39.89 ± 0.46 and 38.61 ± 0.33 for oregano, thyme, lemongrass and mandarin EO, respectively, and after microfluidization these values significantly decreased. Similarly to our results, Salvia-Trujillo et al. (2013) also reported a decrease of WI in nanoemulsions containing EOs and alginate as the aqueous phase after microfluidization. Moreover, we observed significantly different WI of nanoemulsions depending on the EO type. Lemongrass or mandarin nanoemulsions presented significantly lower WI values (27.89 ± 0.08 and 26.77 ± 0.49) than oregano or thyme nanoemulsions (44.50 ± 0.17 and 32.94 ± 0.03). There are few reports about WI in nanoemulsions. To the extent of our knowledge, Salvia-Trujillo et al. (2013) obtained a similar result in the WI value for primary emulsions (54.83) containing lemongrass EO and alginate, and after microfluidization (150 MPa), these values decreased up to 35.71 and 32.63 at 3 and 10 cycles, respectively. They concluded that the number of processing cycles did not affect to a great extent the WI of nanoemulsions. It is known that the emulsion color depends mainly of light scattered, the refractive index of continuous and dispersed phase, oil concentration, droplet concentration and size (McClements, 2002). Fig. 7 shows the changes of WI values during storage time, where significant differences were observed regarding the oil type used in the formulation of nanoemulsions. The color of nanoemulsions containing lemongrass and mandarin remained practically constant during storage time, presenting a transparent appearance (Fig. 1). On other hand, the values of WI for nanoemulsions with oregano and thyme EO increased with storage time. We attributed this change to the increase in average droplet size induced by the aggregation of oil droplets along the time, which was corroborated by visual observations (Fig. 1). In a study carried out by Weiss and McClements (2001), the color of an oil-in-water emulsion changed considerably over time, and they suggest that is related to changes in particle size caused by the growth of individual droplets due mainly to Ostwald ripening phenomenon.

444

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

Fig. 4. Transmission electron microscopy images of essential oil-pectin nanoemulsions produced by microfluidization process (150 MPa and 5 cycles) at time 0 (days). (a) Oregano, (b) Thyme, (c) Lemongrass and (d) Mandarin.

Fig. 5. Examples of droplet sizes measured using a tool of the transmission electron microscope.

3.6. Viscosity Storage time (days) 0

7

14

21

28

35

42

49

56

63

Z potential (mV)

0 -5 -10 -15 -20

-25 Oregano

Thyme

Lemongrass

Mandarin

-30 Fig. 6. Changes in z-potential values of essential oil-pectin nanoemulsions (produced by microfluidization at 150 MPa and 5 cycles) during storage time at 25  C. Data shown are a mean ± standard deviation.

Nowadays, the rheological behavior of emulsions has been of great interest for practical industrial applications. The main factors that influence emulsion viscosity are the dispersed phase volume fraction, the droplet size and droplet charge as well as colloidal interactions (Tadros, 1994). Food hydrocolloids increase the viscosity of aqueous solutions while ionic strength, molecular weight and €k, temperature are some factors that affect their viscosity (Morris, Ko Harding, & Adams, 2010). Pectin is used as gelling, emulsifying and/ or stabilizing agent. In this study, it is used as stabilizer in the formulation of EO-loaded nanoemulsions to enhance their longterm stability. Our results showed that the initial apparent viscosity of the primary emulsions was 48.35 ± 0.22, 40.90 ± 0.41, 46.06 ± 0.24 and 44.10 ± 0.26 mPa s. However, after microfluidization those values dropped down to 21.49 ± 0.37, 22.24 ± 0.46, 23.11 ± 0.28 and 20.01 ± 0.38 mPa s, respectively, producing a reduction of 55, 45, 49, and 54%, respectively. The most remarkable changes in the viscosity of nanoemulsions after microfluidization

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

100

Oregano

Thyme

Lemongrass

during time regardless of the EO type. Bonilla et al. (2012) evaluated the influence of EO type (basil and thyme) on the physical properties of emulsions stabilized with chitosan, found that the incorporation of EOs had a significant effect on the viscosity. Emulsions containing thyme oil were less viscous than that containing basil oil. They attributed these variations to the affinity among chitosan and the droplet surface of oils, affecting the polymer interfacial adsorption, which, in turn, affects the droplet size.

Mandarin

80

WI (%)

445

60 40 20

4. Conclusions 0 0

7

14

21

28

35

42

49

56

63

Storage time (days) Fig. 7. Changes in whiteness index (WI) of essential oil-pectin nanoemulsions (produced by microfluidization at 150 MPa and 5 cycles) during storage time at 25  C. Data shown are a mean ± standard deviation.

25

Viscosity (mPa·s)

20 15 10 5 Oregano

Thyme

Lemongrass

Mandarin

0 0

7

14

21

28

35

42

49

56

63

The results of the current work present useful information for the design of long-term stable nanoemulsions to be used as delivery systems in food products and beverages. The average droplet size of fresh EOepectin nanoemulsions decreased after microfluidization process regardless of the EO type used, being smaller than 50 nm, which was confirmed by TEM. Nanoemulsions containing lemongrass or mandarin EO present a high stability during storage, with a constant droplet size below 100 nm during 56 days as well as absence of creaming. By the contrary, oregano or thyme EO-pectin nanoemulsions were unstable over storage, as seen on the changes in droplet size, droplet size distribution and creaming. This fact may be attributed to Ostwald ripening but also to the lower electrostatic repulsion between droplets due to a lower pectin adsorption on the oil-water interface. The results of the current work present valuable information for the optimal design of long-term stable nanoemulsions to be used as delivery systems in food products. However, information about the antimicrobial activity of nanoemulsions during storage are needed in order to design long-term stable effective antimicrobial delivery systems for food products.

Storage time (days) Fig. 8. Changes in viscosity values of essential oil-pectin nanoemulsions (produced by microfluidization at 150 MPa and 5 cycles) during storage time at 25  C. Data shown are a mean ± standard deviation.

were observed in those containing oregano and mandarin EO (Fig. 8). The decrease of particle size after microfluidization process was accompanied by the decrease of apparent viscosity for all nanoemulsions, fact that can be attributed to the droplet breakup that occurs in turbulent flow in the high shear region where the jets collide and the geometry of the device creates sufficient shear to break the droplets. Also, changes in the molecular conformation of biopolymers after microfluidization can cause the drop of viscosity. In previous studies, microfluidization-induced degradation of polymers, which was attributed to mechanical degradation due to powerful shear, turbulence, high-velocity impaction, highfrequency vibration, instantaneous pressure drop, and cavitation forces generated simultaneously during the treatment (Chen, Huang, Tsai, Tseng, & Hsi, 2011; Liu et al., 2009). In this line, the same behavior has been reported by Salvia-Trujillo et al. (2013), where they attributed the decrease in viscosity values of nanoemulsions to molecular changes in the structure of sodium alginate present in the aqueous phase. Chen et al., (2012), studied the influence of microfluidization process on the rheological properties of high-methoxyl pectin, observing a decrease in the apparent viscosity, average molecular weight and particle size of pectin at more intense treatment conditions, but highlighting that the process had no direct effect on the primary structure of pectin. Therefore, changes in viscosity in our study may be attributed to the loss of pectin molecular weight, which could be due to the breakdown of the polymer chain after microfluidization. The EO type used in the formulation significantly affected the viscosity of nanoemulsions. Moreover, initial viscosity values remained practically constant

Acknowledgments This study was supported by the Ministerio de Ciencia e
n (Spain) throughout the project ‘Improving quality and Innovacio functionality of food products by incorporating lipid nanoparticles into edible coatings’ (AGL2012-35635). Prof. Olga Martín-Belloso
Catalana de Recerca i Estudis Avançats (ICREA) thanks the Institucio
s Guerra for the Academia 2008 Award. Doctoral Student María Ine Rosas thanks the Consejo Nacional de Ciencia y Tecnología (CON
xico, for the scholarship granted number 251520. ACYT), from Me References
s, L., Vargas, M., & Chiralt, A. (2012). Effect of essential oils and Bonilla, J., Atare homogenization conditions on properties of chitosan-based films. Food Hydrocolloids, 26(1), 9e16. Burapapadh, K., Kumpugdee-Vollrath, M., Chantasart, D., & Sriamornsak, P. (2010). Fabrication of pectin-based nanoemulsions loaded with itraconazole for pharmaceutical application. Carbohydrate Polymers, 82(2), 384e393. Burguera, J. L., & Burguera, M. (2012). Analytical applications of emulsions and microemulsions. Talanta, 96, 11e20. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods- a review. International Journal of Food Microbiology, 94(3), 223e253. Chen, R. H., Huang, R., Tsai, L., Tseng, Z., & Hsi, C. (2011). Differences in degradation kinetics for sonolysis, microfluidization and shearing treatments of chitosan. Polymer International, 60, 897e902. Chen, J., Liang, R., Liu, W., Liu, C., Li, T., Tu, Z., et al. (2012). Degradation of highmethoxyl pectin by dynamic high pressure microfluidization and its mechanism. Food Hydrocolloids, 28, 121e129. Dickinson, E., Golding, M., & Povey, M. J. W. (1996). Creaming and flocculation of oilin-water emulsions containing sodium caseinate. Journal of Colloid and Interface Science, 529(185), 515e529. Dickinson, E., & Ritzoulis, C. (2000). Creaming and rheology of oil-in-water emulsions containing sodium dodecyl sulfate and sodium caseinate. Journal of Colloid and Interface Science, 224, 148e154. Henry, J. V. L., Fryer, P. J., Frith, W. J., & Norton, I. T. (2009). Emulsification mechanism and storage instabilities of hydrocarbon-in-water sub-micron emulsions stabilised with Tweens (20 and 80), Brij 96v and sucrose monoesters. Journal of

446

M.I. Guerra-Rosas et al. / Food Hydrocolloids 52 (2016) 438e446

Colloid and Interface Science, 338(1), 201e206. Heurtault, B., Saulnier, P., Pech, B., Proust, J., & Benoit, J. (2003). Physico-chemical stability of colloidal lipid particles. Biomaterials, 24, 4283e4300. Hoeller, S., Sperger, A., & Valenta, C. (2009). Lecithin based nanoemulsions: a comparative study of the influence of non-ionic surfactants and the cationic phytosphingosine on physicochemical behaviour and skin permeation. International Journal of Pharmaceutics, 370(1e2), 181e186. Hsu, J., & Nacu, A. (2003). Behavior of soybean oil-in-water emulsion stabilized by nonionic surfactant. Journal of Colloid and Interface Science, 259, 374e381. Jafari, S. M., He, Y., & Bhandari, B. (2006). Optimization of nano-emulsions production by microfluidization. European Food Research and Technology, 225(5e6), 733e741. Klang, V., Matsko, N. B., Valenta, C., & Hofer, F. (2012). Electron microscopy of nanoemulsions: an essential tool for characterisation and stability assessment. Micron, 43(2e3), 85e103. €blom, J. (2009). Surfactants used in food industry: a review. Journal Ktalova, I., & Sjo of Dispersion Science and Technology, 30, 1363e1383. Liu, L., Fishman, M. L., & Hicks, K. B. (2007). Pectin in controlled drug delivery e a review. Cellulose, 14(1), 15e24. Liu, W., Liu, J., Xie, M., Liu, C., Liu, W., & Wan, J. (2009). Characterization and highpressure microfluidization-induced activation of polyphenoloxidase from Chinese pear (Pyrus pyrifolia nakai). Journal of Agricultural and Food Chemistry, 57, 5376e5380. Lu, W., Zhang, Y., Tan, Y.-Z., Hu, K.-L., Jiang, X.-G., & Fu, S.-K. (2005). Cationic albumin-conjugated pegylated nanoparticles as novel drug carrier for brain delivery. Journal of Controlled Release, 107(3), 428e448. Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B., & Graves, S. M. (2006). Nanoemulsions: formation, structure, and physical properties. Journal of Physics: Condensed Matter, 18, R635eR666. Mayer, S., Weiss, J., & McClements, D. J. (2013). Behavior of vitamin E acetate delivery systems under simulated gastrointestinal conditions: lipid digestion and bioaccessibility of low-energy nanoemulsions. Journal of Colloid and Interface Science, 404, 215e222. McClements, D. J. (2002). Colloidal basis of emulsion color. Current Opinion in Colloid and Interface Science, 7, 451e455. Mirhosseini, H., Tan, C. P., Aghlara, A., Hamid, N. S. A., Yusof, S., & Chern, B. H. (2008). Influence of pectin and CMC on physical stability, turbidity loss rate, cloudiness and flavor release of orange beverage emulsion during storage. Carbohydrate Polymers, 73(1), 83e91. € k, M. S., Harding, S. E., & Adams, G. G. (2010). Polysaccharide drug Morris, G. A., Ko delivery systems based on pectin and chitosan. Biotechnology and Genetic Engineering Reviews, 27, 257e284. Nazarzadeh, E., Anthonypillai, T., & Sajjadi, S. (2013). On the growth mechanisms of nanoemulsions. Journal of Colloid and Interface Science, 397, 154e162.
rez-Espitia, P. J., Du, W.-X., Avena-Bustillos, R. de J., Ferreira-Soares, N. de F., & Pe McHugh, T. H. (2014). Edible films from pectin: Physical-mechanical and antimicrobial properties e A review. Food Hydrocolloids, 35, 287e296. €der, K. (2010). Application of atomic Preetz, C., Hauser, A., Hause, G., Kramer, A., & Ma force microscopy and ultrasonic resonator technology on nanoscale: distinction of nanoemulsions from nanocapsules. European Journal of Pharmaceutical Sciences, 39, 141e151. Qian, C., & McClements, D. J. (2011). Formation of nanoemulsions stabilized by model food-grade emulsifiers using high-pressure homogenization: factors affecting particle size. Food Hydrocolloids, 25(5), 1000e1008.

Rao, J., & McClements, D. J. (2012). Food-grade microemulsions and nanoemulsions: role of oil phase composition on formation and stability. Food Hydrocolloids, 29(2), 326e334. Robins, M. M. (2000). Emulsions- creaming phenomena. Current Opinion in Colloid & Interface Science, 5, 265e272. Sagalowicz, L., & Leser, M. E. (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science, 15(1e2), 61e72. Salvia-Trujillo, L., Rojas-Graü, M. A., Soliva-Fortuny, R., & Martín-Belloso, O. (2013). Effect of processing parameters on physicochemical characteristics of microfluidized lemongrass essential oil-alginate nanoemulsions. Food Hydrocolloids, 30(1), 401e407.
nchez-Gonza
lez, L., Vargas, M., Gonza
lez-Martínez, C., Chiralt, A., & Cha
fer, M. Sa (2011). Use of essential oils in bioactive edible coatings. Food Engineering Reviews, 3, 1e16. Seta, L., Baldino, N., Gabriele, D., Lupi, F. R., & de Cindio, B. (2013). The influence of carrageenan on interfacial properties and short-term stability of milk whey proteins emulsions. Food Hydrocolloids, 32(2), 373e382. Solans, C., Izquierdo, P., Nolla, J., Azemar, N., & Garcia-Celma, M. (2005). Nanoemulsions. Current Opinion in Colloid & Interface Science, 10(3e4), 102e110. Sungthongjeen, S., Sriamornsak, P., Pitaksuteepong, T., Somsiri, A., & Puttipipatkhachorn, S. (2004). Effect of degree of esterification of pectin and calcium amount on drug release from pectin-based matrix tablets. American Asociation of Pharmaceutical Scientists, 5(1), 1e8. Tadros, T. F. (1994). Fundamental principles of emulsion rheology and their applications. Colloids and Surfaces: Physicochemical and Engineering Aspects, 91, 39e55. Tadros, T., Izquierdo, P., Esquena, J., & Solans, C. (2004). Formation and stability of nano-emulsions. Advances in Colloid and Interface Science, 108e109, 303e318. Taylor, P. (1998). Ostwald ripening in emulsions. Advances in Colloid and Interface Science, 75, 107e163. Vallar, S., Houivet, D., Fallah, J. E., Kervadec, D., & Haussonne, J.-M. (1999). Oxide slurries stability and powders dispersion: optimization with zeta potential and rheological measurements. Journal of the European Ceramic Society, 19, 1017e1021.
fer, M., Albors, A., Chiralt, A., & Gonza
lez-Martínez, C. (2008). Vargas, M., Cha Physicochemical and sensory characteristics of yoghurt produced from mixtures of cows' and goats' milk. International Dairy Journal, 18(12), 1146e1152. Weiss, J., & McClements, D. J. (2001). Color changes in hydrocarbon oil-in-water emulsions caused by Ostwald ripening. Journal of Agricultural and Food Chemistry, 49, 4372e4377.
rez, M., Torcello-Go
mez, a., Ga
lvez-Ruíz, M. J., & Martín-Rodríguez, a. Wulff-Pe (2009). Stability of emulsions for parenteral feeding: preparation and characterization of o/w nanoemulsions with natural oils and Pluronic f68 as surfactant. Food Hydrocolloids, 23(4), 1096e1102. Yao, X., Wang, N., Fang, Y., Phillips, G. O., Jiang, F., Hu, J., et al. (2013). Impact of surfactants on the lipase digestibility of gum arabic-stabilized O/W emulsions. Food Hydrocolloids, 33(2), 393e401. Ye, A., & Singh, H. (2006). Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides. Food Hydrocolloids, 20, 269e276. Ziani, K., Chang, Y., Mclandsborough, L., & McClements, D. J. (2011). Influence of surfactant charge on antimicrobial efficacy of surfactant-stabilized thyme oil nanoemulsions. Journal of Agricultural and Food Chemistry, 59, 6247e6255.