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submicron emulsions containing fenugreek gum
Accepted Manuscript Stability and physical properties of model macro- and nano/submicron emulsions containing fenugreek gum Olga Kaltsa, Neolea Spiliopoulou, Stavros Yanniotis, Ioanna Mandala PII:
S0268-005X(16)30275-2
DOI:
10.1016/j.foodhyd.2016.06.025
Reference:
FOOHYD 3479
To appear in:
Food Hydrocolloids
Received Date: 31 March 2016 Revised Date:
25 May 2016
Accepted Date: 20 June 2016
Please cite this article as: Kaltsa, O., Spiliopoulou, N., Yanniotis, S., Mandala, I., Stability and physical properties of model macro- and nano/submicron emulsions containing fenugreek gum, Food Hydrocolloids (2016), doi: 10.1016/j.foodhyd.2016.06.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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2ο macroemulsion
Ultrasounds 1ο nanoemulsion
2ο nanoemulsion
+ Fenugreek Gum
5 month storage
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1ο macroemulsion
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+ Fenugreek Gum
ACCEPTED MANUSCRIPT Stability and physical properties of model macro- and nano/submicron emulsions containing fenugreek gum
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Olga Kaltsa, Neolea Spiliopoulou, Stavros Yanniotis, and Ioanna Mandala1
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Food Science & Nutrition Dept., Agricultural University of Athens, Iera Odos 75,
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11855, Athens, Greece.
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Abstract
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Nanoemulsions were made in two steps. Firstly, a less viscous, primary O/W
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emulsion was prepared using ultrasounds. In those emulsions, only whey protein
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isolate was used as an emulsifier, whereas the oily phase was olive oil. A stabilizer,
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fenugreek gum, was incorporated afterwards in the primary formulations resulting in
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the secondary emulsions of increased viscosity. Oil amount changed and its decrease
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from 20 to 5 % wt led to a reduced average droplet size and polydispersity index
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values in the primary emulsions. Nanoemulsions with the lowest polydispersity were
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produced at a high sonication time treatment of 12 min. Their droplet size was ~200
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nm. They showed increased viscoelastic properties and consistency leading to
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significantly increased stability compared to their coarse secondary emulsions
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produced by high shear mixing. Even the formulations with the lowest olive oil
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content investigated, 2.5% wt, presented an improved stability during a long-term
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storage of six months.
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Corresponding author: Ioanna Mandala ([email protected]), 75 Iera Odos, Votanikos, Athens, Tel.:+30 2105294692, Fax:+30 210 5294697
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ACCEPTED MANUSCRIPT 1. Introduction
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Emulsions can have very fine droplets of ten to a few hundred nanometers in
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diameter, namely nanoemulsions. They are designed to have a high kinetic stability
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considering that creaming or sedimentation velocity is proportional to the square of
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the particle size. Additionally, they have been praised for increased bioavailability of
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several bioactive compounds (McClements, 2005; Sonneville-Aubrun, Simonnet, &
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L’Alloret, 2004; Tadros, Izquierdo, Esquena, & Solans, 2004).
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In our study, virgin olive oil was used to form an oil in water emulsion. This is
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considered significant as many strategies and actions have been globally undertaken
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by several initiatives and authorities to overcome obesity pandemic, known as
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“globesity”. In particular, the World Health Organization suggestions towards fat
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include a consumption shift from saturated to unsaturated fats and the restriction of
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energy intake from fat (WHO, 2015).
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Fenugreek (Trigonella foenum-graecum L.) seeds are generally used as a spice but
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they also have medicinal properties. They contain 45% to 60% carbohydrate (mainly
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galactomannan), 6% to 10% lipid (mainly polyunsaturated fatty acids), and 20% to
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30% protein (4-hydroxyisoleucine being one of the major amino acids) (Rao,
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Sesikeran, Rao, Naidu, Rao, & Ramacjandran, 1996; Raghuram, Sharma, &
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Sivakumar, 1994). Over the last decade, the use of fenugreek gum has been increased
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in many food products, because of its considerable thickening, stabilizing, and
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emulsifying properties (Işıklı & Karababa, 2005). Additionally, fenugreek gum
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contains a low amount of protein. These proteinaceous moieties, at concentration as
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low as 0.1 wt %, impart an amphiphilic property to the gum, which can then act
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somehow like an emulsifier (Brummer, Cui & Wang, 2003; Youssef, Wang, Cui, &
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Barbut, 2009).
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In a previous study (Kaltsa,Yanniotis & Mandala, 2016) we have shown that
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fenugreek gum fractions with high galactomannan content (>80 %wt) may exhibit
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similar or improved stability properties in whey protein emulsions compared to other
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commercial galactomannans. However, when a high-energy homogenization process
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is used, such as ultrasonication, a severe degradation of commonly used
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polysaccharide stabilizers takes place, owed to extreme shearing. Hence,
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polysaccharides’ addition in the initial formulation is not suggested, as their
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degradation can reduce the viscosity of the continuous phase enhancing the
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ACCEPTED MANUSCRIPT destabilization process (Kaltsa, Michon, Yanniotis & Mandala, 2013; Kaltsa et al.,
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2016; Tiwari, Muthukumarappan, O'Donnell, & Cullen, 2010). Additionally,
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stabilizers can hinder the efficacy of the homogenization process. They increase the
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viscosity, which hinders the propagation of soundwaves, increases the cavitation
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threshold to higher amplitudes and reduces the shearing turbulence (Wooster,
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Golding, & Sanguansri, 2008).
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The stability of whey prrotein nanoemulsions has been the topic of many studies
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lately (Adjonu, Doran, Torley, & Agboola, 2014a, b; Ali, Mekhloufi, Huang, &
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Agnely 2016; Ozturk, Argin, Ozilgen, & McClements, 2015; Teo et al., 2016; Zeeb,
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Herz, McClements, & Weiss, 2015). However, to our best of knowledge, there is no
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available information regarding nanoemulsion models containing fenugreek gum.
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The objective of this study is the investigation of nanoemulsion formation by
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changing the oil fraction, and ultrasonication processing time. In general low oil
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fractions (<10 %) favor the formation of nanoemulsions below 200 nm, while higher
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ones promote the formation of larger ones (Leong, Wooster, Kentish, & Ashokkumar,
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2009; Tabibiazar et al., 2015). Extended sonication time may also be useful in order to
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reduce the mean droplet diameter of nanoemulsions as in a longer process there is
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more time for the protein to orientate on the oil interface (Jafari Ηe, & Bhandari,
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2007). The stability of the final formulations (secondary model emulsions) produced
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by further dilution with fenugreek gum was evaluated and comparisons were made
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between nanoemulsions and their macroemulsion homologues produced by high-
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speed mixing.
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2. Materials and methods
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2.1. Materials
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Whey protein isolate (WPI) Lacprodan DI-9224 was kindly provided by Arla (Arla
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Foods Ingredients, Amba-Denmark). The composition of WPI powder in protein as
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stated by the manufacturer was protein 92 ± 2 %, and maximum amounts of fat 0.2 %,
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ash 4.5 % and lactose 0.2 %. Fenugreek gum powder Fenulife® was a kind gift from
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Frutarom(Londerzeel, Belgium) (moisture content 6.3 %, soluble fiber content 83.2
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%, mostly composed of galactomannan according to manufacturer and 3.2 % protein
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calculated by Dumas method (Kaltsa, Yanniotis & Mandala, 2016)). Virgin olive oil
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Altis (Elais Unilever, Greece) was purchased from a local store. Sodium azide was
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from Sigma–Aldrich.
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2.2. Solution preparation
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Whey protein stock solutions (10 g in deionized water in total weight 95, 90 or 80 g)
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were prepared by agitation with a magnetic stirrer for 90 min. Fenugreek gum (FGF)
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solutions 1, 1.5 and 2 %wt in deionized water were prepared by hot stirring in a water
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bath at 90 oC for 90 min. Thereafter, solutions were kept overnight at 5 oC to ensure
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complete hydration. Sodium azide 0.02 %wt was added to the aqueous solutions as an
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antimicrobial agent.
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2.3. Coarse emulsion preparation and screening
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Oil-in-water primary macro-emulsions were prepared by homogenizing 5, 10 or 20 g
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of oil phase with 95, 90 or 80 g of the WPI aqueous solutions accordingly. The initial
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step in preparing macro-emulsions involved the production of a coarse premix
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emulsion by homogenizing the oil and the aqueous phases using an Ultra Turrax (T25
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basic, Janke & Kunkel IKA Labortechnik, Staufen, Germany) for 2 min (13.500
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RPM) at room temperature. The coarse emulsions were further diluted with fenugreek
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gum solutions (2 %wt) in a ratio of 1:1 by weight. Ingredients were further mixed
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using a magnetic stirrer for 1 min in order to avoid recoalescence of the droplets and
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to yield to the secondary coarse emulsions. Emulsion preparation was conducted in
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neutral conditions in order to take advantage of the higher solubility of the protein.
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The pH of the resulting macro- and nano-emulsions was then adjusted to 3.8, which is
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typical for acidic food emulsions, with a few drops of glacial acetic acid.
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Nanoemulsions were produced by further homogenizing 10 ml of the primary coarse
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premixes with an ultrasonic homogenizer model Sonopuls 3200 (Bandelin Electronic
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Gmbh & Co, Berlin) equipped with a 3 mm in diameter titanium probe (MS 73). The
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tip was immersed 10 mm from the surface of the sample which was placed in a double
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jacketed flow-through vessel (model DG-3). The temperature was maintained at 30 ±
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1oC by circulating cold water with a pump. The amplitude was set at 40%. It was
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noticed that amplitude values higher than 40 % led to increased fluctuations of the
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samples in the vessel, which caused sample spilling. The processing time ranged
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between 1 up to 12 min. The experimental data at time intervals of 1, 2, 4, 8 and 12
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min showed that the nominal ultrasonic energy input (kJ), as a function of time,
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ACCEPTED MANUSCRIPT followed the following linear equation: Energy (kJ) = 1.88 x Time + 0.1524 (R2=1).
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The emulsification time, which resulted in both minimum droplet sizes and
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polydispersity index (PDI) for all oil concentrations used, was chosen for the
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incorporation of nanoemulsions in 2 %wt fenugreek solutions as mentioned above for
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macro-emulsions. All samples were prepared in triplicate.
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2.4. Droplet size measurement
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Droplet size distributions of the primary coarse emulsions were determined using a
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Mastersizer 2000 (Malvern Instruments, Malvern, UK). Emulsion droplets were
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characterized under high dilution conditions by dispersing the samples in deionized
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water. The refractive indices of water and olive oil were taken as 1.330 and 1.467,
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respectively, and the Mie theory was used for the analysis (Paximada, Tsouko,
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Kopsahelis, Koutinas, & Mandala, 2014). Average droplet sizes were characterized in
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terms of the volume mean diameter D50 and polydispersity (Span). All measurements
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were made at ambient temperature on three separately prepared samples.
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The mean droplet size (z-average diameter) and the polydispersity index (PDI) of
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nanoemulsions were measured using a dynamic light scattering instrument (Zetasizer
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Nano ZS, Malvern Instruments, Malvern, UK). Measurements were performed at
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25 °C in triplicate. Prior to measurement, the nanoemulsions were diluted (1:500)
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with deionized water to avoid multiple scattering during measurements.
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2.5. Emulsion stability
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The storage stability (at 5 οC) of macro- and nanoemulsions was determined by
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obtaining backscattering profiles with a Turbiscan 2000 device (Turbiscan 2000,
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Formulaction, Toulouse). For unstable macro emulsions the serum percentage was
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calculated (Zinoviadou, Scholten, Moschakis, & Biliaderis, 2012). In stable samples
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(macro- and nano/submicron emulsions), the average value of the backscattering (BS)
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measurement at the middle zone of the cell (between 30 and 40 mm of sample height)
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has been plotted as a function of ageing time and BS variation (dBS) during 10 days
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of storage. Calculations were conducted as described in Kaltsa et al. (2013).
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2.6. Emulsion rheology
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Viscosity measurements were performed with a hybrid rheometer DHR (TA
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Instruments, New Castle, DE, US) equipped with plate-plate geometry (upper plate
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ACCEPTED MANUSCRIPT diameter 50 mm, gap 1 mm) to obtain flow curves between 0.1 to 1000 s-1 at 25 ±
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0.01 oC by circulating water from a peltier. The total measurement time was 900 s. All
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measurements were performed in triplicate and data reported as average and standard
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deviations.
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The linear viscoelastic region (LVR) was assessed at 6.28 rad/s (= 1 Hz) by strain
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sweep experiments; for all samples a constant deformation of γ = 0.1 % was used,
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which was within the linear viscoelastic region of all the samples. Deviation from the
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linear viscoelastic region occurs when the sample starts to permanently deform,
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implying the destruction of the transient network structure. Small deformation
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oscillatory measurements were performed for the evaluation of the viscoelastic
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properties, G′ (storage modulus), G″ (loss modulus), and tanδ (G″/G′) (Steffe,
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1996; Metzger, 2006), over the frequency range of 0.1–100 rad/s at 25 °C.
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3. Results and discussion
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3.1. Droplet size
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The droplet mean diameter (D50) of primary coarse emulsions ranged between
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9.48 and 13.76 µm (Table 1). Although emulsion diameter usually increases when
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increasing oil concentration (Christensen et al., 2001), only a slight change in the
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mean diameter was observed for samples containing 20 %wt olive. Nevertheless, no
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significant differences were observed compared to emulsions containing lower
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amounts of olive oil (p>0.05). No significant differences were observed either
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considering the polydispersity of samples since Span values ranged between 1.46 up
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to 2.48 (Table 1). This allows us to assume that the emulsification time and protein
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isolate concentration used, were sufficient to equally minimize the droplet size in all
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formulations examined regardless of the oil content.
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It is well known that WPI, as a protein, is a surface-active molecule that has the
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tendency to adsorb at interfaces and undergoes structural rearrangement. However, its
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solubility and emulsifying properties depend on pH. As mentioned above, emulsion
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preparation was conducted at neutral conditions in order to take advantage of higher
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solubility of the protein. In our study, the whey protein used, presented a minimum of
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93.6 % solubility (data not shown) at pH 5, which is near the typical value of whey
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proteins’ pI ~ 4.5 (Pelegrine and Gasparetto, 2005) and should be mentioned that only
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undenatured whey proteins remain soluble throughout all pΗ range
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ACCEPTED MANUSCRIPT Αt neutral pH values, oil droplets with whey protein absorbed at the interface possess
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a negative charge and are strongly repelled. On the contrary, at pH values near pI, the
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surface charge diminishes and repulsive forces are weakened, leading to enhanced
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attraction of the droplets. At even lower pH whey proteins take on a net positive
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electrical charge. They can then interact with charge-carrying polysaccharides (e.g.
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pectin, xanthan, carrageenan, chitosan) and extensive interactions may take place
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either increasing or decreasing the stability of the emulsion. In our case, the reduction
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of pH did not induce any instability, as fenugreek gum, like all other galactomannans,
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does not carry any electrostatic charge to interact with the positively charged WPI on
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droplet interface. However, proteinaceous moieties in fenugreek gums may impart an
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amphiphilic property to the gum, which enables it to act somehow like an
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emulsifier (Brummer et al., 2003; Youssef et al., 2009; Kaltsa et al., 2016).
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Primary coarse emulsions were subjected to sonication in order to achieve further
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droplet size reduction. As shown in Fig. 2a, a sharp decrease in the droplet size was
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achieved within 1 min of sonication, hence the z-average diameter ranged between
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323 - 288 nm depending on the oil content. Thereafter, the droplet size was
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moderately reduced at increased sonication times up to 12 min. In more detail, the z-
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average diameter of samples containing 5 and 10 %wt oil did not show any
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considerable droplet reduction after 1 or 2 min of sonication accordingly, whereas a
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longer time of 8 min was necessary that samples containing 20 % wt oil reach a
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droplet size plateau.
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Regarding the polydispersity of sonicated emulsions, it is shown that the PDI
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decreased, when the sonication processing time increases (Fig. 2b). Although the PDI
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values for samples containing 5 and 10 %wt oil, was 0.434 and 0.51 respectively after
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1 min of sonication whereas the PDI values were considerably higher in samples
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containing 20% olive oil. More specifically, emulsions containing 20 %wt olive oil
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were characterized by
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polydispersity of the droplets (Tiwari, Hihara, & Rawlins, 2014). However, results
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should be treated with caution since DLS measurements can be considered reliable
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when values of PDI <0.5 are obtained.
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At low oil fractions 5 and 10 %, PDI values were unvarying after 4 or 8 min
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accordingly. However, PDI values reduction under sonication time increase was
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remarkable in samples containing 20% olive oil. Actually, the lowest PDI value was
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observed at 12 min of sonication in emulsions containing 20% of olive oil.
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PDI values greater than 0.9, indicative of the strong
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ACCEPTED MANUSCRIPT Considering the above findings, a sonication time of 12 min was selected in order to
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further add the FGF gum, as that time resulted in the lowest PDI values for all
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samples produced.
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Nanoemulsions with an average size droplets of 207 nm and a narrow polydispersity
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(~ 0.2) at oil fraction 5% wt can be seen (Fig. 2a, b). This size is slightly higher than
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that reported by Adjonu et al. (2014a), who prepared WPI nanoemulsions of 160 nm,
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which could be attributed to slightly lower oil fraction used (4 %wt). Lower droplet
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sizes reaching 75 nm have been referred by using bovine serum albumin (BSA) in the
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presence of polyethylene-glycol (PEG) (Tabibiazar et al., 2015).
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An increase in the oil fraction to 10 or 20% led to higher droplet sizes (~280 nm) and
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PDI values (~0.38), although no significant differences were observed after 12 min of
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sonication (p>0.05) (Table 1).
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3.2. Screening of FGF concentration for secondary coarse emulsion preparation
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Initially, a screening procedure was conducted in order to assess the FGF
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concentration required to obtain stable secondary coarse emulsions containing 2.5, 5,
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or 10 %wt olive oil in the final formulation. The curves of creaming (% Serum)
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versus storage time of coarse emulsions containing 0.5 up to 1 %wt FGF are shown in
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Fig. 3.
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The oil phase content significantly affected the serum percentage values (p<0.05). As
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can be seen the stability of emulsions was improved by increasing the oil content from
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2.5 to 10 %wt (Fig. 3a, b,c).
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For emulsions containing 0.5 %wt FGF the serum percentage decreased from 71.3 to
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~ 9.5 % when the oil content changed from 2.5% to 10%wt, while for those
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containing 0.75 %wt FGF, a reduction from 42.2 to 3.2 % was noticed for the same
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oil content change. Stability improvement by increasing the oil content is well known.
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It is associated to the increase in packing fraction of oil droplets, which leads to
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enhanced interdroplet interactions and viscosity and to more structured emulsions
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(Dickinson & Golding, 1997; Sun & Gunasekaran, 2009; Nikiforidis, Biliaderis, &
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Kiosseoglou, 2012). However, the oil concentrations used are far from the droplet
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close packing limits. In fact, creaming becomes inhibited altogether when the oil
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volume fraction φ is as high that the droplets become close-packed, which is φ ~ 0.6
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for monodisperse spheres (Dickinson, 2009).
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stability and prevent creaming through viscosity modification of the aqueous
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continuous phase (Dickinson, 2003). In our case, the addition of 1 %wt FGF resulted
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in the formation of stable coarse emulsions, which did not show any phase separation
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during 10 days of storage. Therefore, this concentration was used in subsequent
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formulations and comparisons were made between coarse and nanoemulsion
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emulsions. Huang, Kakuda, & Cui (2001), who compared the stability of emulsions
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containing different hydrocolloids, have also shown that long term stability without
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phase separation could be achieved by the use of higher concentration of fenugreek,
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guar and locust gums (1-1.5 %wt), attributed to a droplet movement restriction caused
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by the continuous phase viscosity increase.
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3.3. Emulsion rheological properties
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The Power law model was adopted to describe the viscosity properties of coarse and
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nanoemulsion analogues, as the same model was applied to describe the shear
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thinning behavior of extruded fenugreek gum solutions (Chang & Cui, 2011).
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Table 2 summarizes the estimated consistency (k) and flow behavior (n) values of
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prepared secondary coarse and nanoemulsions containing 1 %wt FGF and various
265
amounts of olive oil. All model emulsions prepared were highly pseudoplastic with
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flow coefficient (n) values varying from 0.29 to 0.35.
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Consistency and flow behavior values were both influenced by the oil content and the
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preparation method used, but the greatest changes were noticed at 10% oil. At 2.5% or
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5% oil content differences among the samples were not significant in most cases
270
(Table 2).
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Considering the coarse emulsions, an increase of oil content from 2.5 to 10 %wt
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increased the consistency of the samples as well, from ~7.4 to 11.7 Pa-sn. This
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change of the oil fraction causes an upward shift of the flow curves to higher
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viscosities, attributed to the increased packing of the oil droplets. This leads to
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enhanced droplet interactions, thus to more viscous emulsions (Sun & Gunasekaran,
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2009). Significant differences were observed among all coarse emulsions regardless
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of their oil content (p<0.05). Nevertheless, this was also observed in nano-emulsions,
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for which, k values increased from 8.59 to 15.69 Pa-sn. The decrease of the droplet
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size within the submicron scale resulted in an increase of the k values by 16 up to 34
280
% depending on oil concentration. However, it may be noticed that no significant
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ACCEPTED MANUSCRIPT differences are found between the viscosity values (k) of submicron emulsions
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containing 2.5 and 5 %wt olive oil (p>0.05).
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The oil fraction increase also led to an enhancement of the pseudoplastic character in
284
both coarse and nano-emulsions, as evidenced by the decrease in flow behavior values
285
(n). Flow behavior values ranged between 0.333 – 0.352 and 0.288 – 0.324 for coarse
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and nanoemulsions respectively. Similar values were reported for 1 %wt fenugreek
287
solutions by Wu et al. (2009), indicating the predominant role of fenugreek on
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emulsion pseudoplasticity, as found in a previous study (Kaltsa et al., 2016).
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Nanoemulsions were more pseudoplastic in all cases, regardless of the oil
290
concentration (p<0.05).
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Dynamic oscillatory shear tests were used to characterize the viscoelastic properties of
292
coarse and nano-emulsions containing 1 %wt FGF. The storage (G’) and loss (G”)
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modulii measured, are shown in Fig. 4(a-c). Loss tangent (tanδ) was also used to
294
assess the elastic or viscous character predominance in the samples as shown in the
295
same figure.
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Overall, samples exhibited frequency-dependent storage and loss modulus values,
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which increased during frequency ramp from 0.1 to 100 rad/s. In all emulsions, G”
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was dominant over G’ in low frequency region, thereby showing a liquid like
299
behavior. This implies that the most of the input energy cannot be stored at this low
300
frequency region, thus it dissipates through viscous flow. At higher frequencies
301
(approximately > 1 rad/s) a cross-over occurs and a solid-like behavior is observed for
302
all emulsion preparations (G’>G”). Additionally, the crossover point of the modulii is
303
shifted at lower frequencies in the viscoelastic spectra of nanoemulsions.
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Entanglements of macromolecular solutions can result in such behavior according to
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Ross-Murphy (1984). Moreover, when G’ is higher than G’’ over most of the
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frequency range investigated, the formation of a weak three-dimensional network of
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ordered chain segments can be evident. The same behavior was observed in
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nanoemulsions of higher oil content (Fig. 4c). However, as the tanδ values were
309
higher than 0.3 over the entire frequency range (Fig. 4), emulsions maintain a viscous
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character (Mandala et al., 2004). Only for a tanδ > 0.1, a weak gel behavior is
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ascribed, whereas a strong gel is evident at tanδ values less than 0.1 (Wang & Cui,
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2005). These findings can be explained by the fact that galactomannans, even at low
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concentrations, are capable of forming highly viscous solutions due to their high water
314
binding capacity. Galactomannans, such as fenugreek and guar gum, do not form gels
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subzero
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temperatures (Dea et al., 1977) or a long storage are required to form gels
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(Richardson, Clark, Russell, Aymard, & Norton, 1999). According to Wu, Cui, Eskin
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& Goff (2009) fenugreek gum solutions with concentration ranging between 0.5 -2
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%wt present a liquid like behavior at lower frequencies (G”>G’) and a solid-like at
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higher ones. Moreover, in another study, the mechanical spectra of 1 %wt fenugreek
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gum aqueous dispersions showed a change of behavior depending on the molecular
322
weight. Fenugreek gum fractions with higher molecular weight (Mw≥ 18.3x105
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g/mol) showed a concentrated polymer solution pattern, exhibiting modulus crossover
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(G’>G’’) at high angular frequencies, as in our case, whereas those of lower Mw
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presented a dilute polymer solution pattern, as evidenced by modulii values (G’’>G’)
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and absence of crossover (Funami et al., 2008).
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The droplet packing caused by oil fraction increase and sonication resulted in
328
emulsion thickening as evidenced by the enhancement of G’ and G’’ values (Sun &
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Gunasekaran, 2009).
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At the same time, a decrease of loss tangent occurred, revealing the increase of the
331
solid like behavior. Minor differences in the storage, loss modulus and tanδ were
332
detected between formulations with low amounts of oil (2.5 and 5 %wt). Obviously,
333
findings of dynamic properties are in-line with those obtained in respect with the flow
334
behavior of the emulsions.
335
3.4. Stability of coarse and nano- emulsions containing 1 %wt FGF
336
Coarse and nanoemulsion formulations at 1 %wt FGF did not exhibit any phase
337
separation within the period tested (10 days, 5οC), as back-scattering values were
338
constant during storage (Fig. 5). According to Mie theory the back scattering intensity
339
increases by a decrease of the particle size of the droplets, when their diameter is
340
greater than 500 nm while the opposite applies for those of smaller size e.g.
341
nanoemulsions. It can be noticed that the initial BS intensity values are affected by oil
342
content and emulsification method. According to several studies BS intensity is a
343
value influenced by the size and density of the oil particles in emulsions. It is
344
enhanced by the increase of the oil droplet number and by decreasing their size or the
345
size of the flocs present (Palazolo, Sorgentini, & Wagner, 2004 and 2005). In our case
346
the increase of the oil fraction from 2.5 to 10 %wt increased the initial BS values of
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ACCEPTED MANUSCRIPT coarse emulsions from 40.3 to 61.6 % and those of nanoemulsions from 45.6 to 71.8
348
%.
349
Concerning storage time, in coarse emulsions containing 5 and 10 %wt oil, a
350
reduction of approximately ~4 % in BS (dBS) was noticed within 10 days of storage,
351
while it reached a value of 11.4 % decrease in samples containing 2.5 %wt oil (Fig.
352
5c). The BS change (dBS) was lower in nanoemulsions and ranged between ~ 1-1.9
353
%, hence an improvement in the stability in terms of the coalescence-flocculation was
354
achieved, attributed to droplet size reduction and thickening of the samples as
355
discussed above.
356
Normally, an increase in BS should have been noticed with storage time, in the case
357
of nanoemulsions, as a consequence of coalescence or flocculation phenomena
358
occurring during storage as previously observed in other studies (Porras, Solans,
359
González, & Gutiérrez, 2008; Wulff-Pérez, Torcello-Gómez, Gálvez-Ruíz, & Martín-
360
Rodríguez, 2009). In our case, the reason for a back scattering decrease could be
361
explained by the fact that nanoemulsions may have undergone flocculation during the
362
incorportation of FGF solution and subsequent pH adjustment from 6.5 to 3.8 passing
363
through the isoelectric point of proteins. The net negative charge that the oil droplets
364
posse at high pH values is suppressed by the decrease of pH values near the pI of the
365
protein (4.8), thus a few droplets of an average diameter 200 - 300 nm could easily
366
form flocs, the size of which could be detected by the Turbiscan wave length.
367
Considering a long storage of five months, as demonstrated in Fig. 6 and 7, coarse
368
emulsions prepared with 2.5 %wt oil were the least stable, since phase separation
369
(serum) occurred as evidenced by a sharp BS decrease near zero values at the bottom
370
of the tube (Fig. 6). Phase separation of that sample is also shown in Fig. 7. For the
371
rest of the samples, BS decreased gradually along
372
coalescence-flocculation phenomena indicated by higher BS values on day 0 (purple
373
line) and lower ones one day 150 (red line).
374
4. Conclusions
375
Low oil content model emulsions were developed and stabilized by FGF addition.
376
Stable coarse formulations were achieved upon the addition of 1 wt % FGF in all
377
cases of oil content (2.5, 5 or 10 %wt). Emulsion stability was further ameliorated by
378
preparing nano/submicron emulsions by ultrasonic homogenization. Nanoemulsions
379
with the smallest droplet diameter (~200 nm) and PDI (~0.2) were produced at the
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the sample tube due to
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ACCEPTED MANUSCRIPT lowest oil content after 12 min of sonication. Droplet size reduction in the
381
nano/submicron scale influenced the viscoelastic properties of the emulsions, as more
382
structured and viscous samples were obtained. All nano/submicron emulsions
383
exhibited higher stability during storage compared to coarse models due to a droplet
384
size reduction and their enhanced viscoelastic properties. Our results demonstrate that
385
the presence of nanosized droplets is mostly beneficial in the case of producing very
386
low oil content (2.5 % wt) model emulsions, as long term stability is still evident.
387
Acknowledgements
388
This research has been co-financed by the European Union (European Social Fund –
389
ESF) and Greek national funds through the Operational Program “Education and
390
Lifelong Learning” of the National Strategic Reference Framework (NSRF) –
391
Research Funding Program: Heracleitus II. Investing in knowledge society through
392
the European Social Fund.
393
The authors would like to thank Frutarom Belgium N.V. for kindly donating
394
FenuLife® extract.
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Figure 1. Volume weighted size distributions of primary emulsions as affected by oil
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concentration.
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Figure 2. Droplet size (a) and polydispersity index (PDI) (b) of emulsions as a
537
function of sonication time and oil concentration. Diamonds correspond to 5 %,
538
squares 10 % and triangles to 20 % oil.
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Figure 3. Stability of coarse emulsions containing as affected by oil (diamonds, 2.5
540
%wt, squares 5 %wt and triangles 10 %wt) and FGF concentration (open symbols: 0.5
541
%wt, closed symbols: 0.75 %wt and “x”, all other emulsions containing 1 %wt).
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Different letters indicate significant differences among samples (p<0.05).
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Figure 4. Effect of oil content on storage (G’, closed symbols) and loss modulus (G”,
544
open symbols) of emulsions containing a) 2.5 %wt (diamonds), b) 5 %wt (squares)
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and c) 10 %wt (triangles) olive oil. Black symbols indicate coarse emulsions and red
546
ones nanoemulsions. Black circles correspond to loss tangent (tanδ) of course
547
emulsions and red circles of nanoemulsions
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Figure 5. Stability of emulsions containing 2.5 (diamonds), 5 (squares) and 10 %wt
549
oil (triangles): a) Coarse emulsions, b) Nanoemulsions and c) Back scattering
550
variation (dBS) after 10 days of storage (5 oC).a-d Different letters indicate significant
551
differences among samples (p<0.05).
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Figure 6. Back scattering profiles of coarse and nano- emulsions containing 1 %wt
553
FGF during storage (5 oC).
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Figure 7. Stability of coarse and nano- emulsions containing 1 %wt FGF after 5
555
months of storage (5 oC).
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Table 1. Droplet size (volume median diameter (D50) and z-average diameter) and
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polydispersity (Span and PDI) of primary emulsions as affected by olive oil
559
concentration and emulsification method applied.
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Table 2. Estimated consistency (k) and flow behavior (n) values of coarse and nano-
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Figure 1 7
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565
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ACCEPTED MANUSCRIPT 566
Figure 3 100
80
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Figure 5 100
80
Back scattering (%)
60
40
60
12 Nanoemulsions 10
40
2
4
6 8 Storage days
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8 c
6 bc
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Oil content (%wt)
10
80%
80%
60%
60%
60%
40%
40%
40%
20%
20%
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0mm
0%
50mm
0d
EP
Back Scattering 100% 80% 60% 40% 20% 0% 0mm
150d
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0% 50mm
150d
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Back Scattering 100%
152d
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Back Scattering
80%
80%
60%
60%
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40%
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0% 150d
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Back Scattering 100%
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ACCEPTED MANUSCRIPT Figure 7 2.5 % wt
5 % wt
10 % wt
Coarse emulsion Nanoemulsion
Coarse emulsion Nanoemulsion
Coarse emulsion Nanoemulsion
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577 578
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Table 1 Oil concentration (%wt) 5
10
20
D50 (µm)
9.73a ± 3.3
9.48a ± 4.09
13.76a ± 3.96
Span (-)
1.81a ± 0.23
2.68a ± 1.08
1.46a ± 0.54
207.3a ± 1.0
288.7b ± 17.6
267.3b ± 8.5
0.219a ± 0.006
0.381b ± 0.021
0.386b ± 0.009
Nano- emulsions Z-average diameter (nm)
581
a-b
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PDI (-)
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Different superscripts per parameter indicate significant differences among samples (p<0.05).
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Table 2 k (Pa-sn)
n (-)
2.5
7.40a ±0.45
0.352d ±0.006
5
8.31b ± 0.32
0.343d ± 0.011
10
11.72c ±1.05
0.333c ±0.003
2.5
8.59b ±0.71
5
9.09b ±0.35
10
15.69d ±0.24
Oil content (%wt)
584
a-d
585
2
0.324bc ±0.006 0.320b ±0.001 0.288a ±0.012
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Nanoemulsions
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ACCEPTED MANUSCRIPT Highlights •
Low oil content macro and nanoemulsion models were prepared containing fenugreek gum
•
Droplet size decrease enhanced the rheological properties of nanoemulsions
•
Nanosized droplets are mostly appreciated for emulsions with the lowest oil
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S0268-005X(16)30275-2
DOI:
10.1016/j.foodhyd.2016.06.025
Reference:
FOOHYD 3479
To appear in:
Food Hydrocolloids
Received Date: 31 March 2016 Revised Date:
25 May 2016
Accepted Date: 20 June 2016
Please cite this article as: Kaltsa, O., Spiliopoulou, N., Yanniotis, S., Mandala, I., Stability and physical properties of model macro- and nano/submicron emulsions containing fenugreek gum, Food Hydrocolloids (2016), doi: 10.1016/j.foodhyd.2016.06.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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2ο macroemulsion
Ultrasounds 1ο nanoemulsion
2ο nanoemulsion
+ Fenugreek Gum
5 month storage
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1ο macroemulsion
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+ Fenugreek Gum
ACCEPTED MANUSCRIPT Stability and physical properties of model macro- and nano/submicron emulsions containing fenugreek gum
1 2 3
Olga Kaltsa, Neolea Spiliopoulou, Stavros Yanniotis, and Ioanna Mandala1
5
Food Science & Nutrition Dept., Agricultural University of Athens, Iera Odos 75,
6
11855, Athens, Greece.
7
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Abstract
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Nanoemulsions were made in two steps. Firstly, a less viscous, primary O/W
10
emulsion was prepared using ultrasounds. In those emulsions, only whey protein
11
isolate was used as an emulsifier, whereas the oily phase was olive oil. A stabilizer,
12
fenugreek gum, was incorporated afterwards in the primary formulations resulting in
13
the secondary emulsions of increased viscosity. Oil amount changed and its decrease
14
from 20 to 5 % wt led to a reduced average droplet size and polydispersity index
15
values in the primary emulsions. Nanoemulsions with the lowest polydispersity were
16
produced at a high sonication time treatment of 12 min. Their droplet size was ~200
17
nm. They showed increased viscoelastic properties and consistency leading to
18
significantly increased stability compared to their coarse secondary emulsions
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produced by high shear mixing. Even the formulations with the lowest olive oil
20
content investigated, 2.5% wt, presented an improved stability during a long-term
21
storage of six months.
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Corresponding author: Ioanna Mandala ([email protected]), 75 Iera Odos, Votanikos, Athens, Tel.:+30 2105294692, Fax:+30 210 5294697
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ACCEPTED MANUSCRIPT 1. Introduction
23
Emulsions can have very fine droplets of ten to a few hundred nanometers in
24
diameter, namely nanoemulsions. They are designed to have a high kinetic stability
25
considering that creaming or sedimentation velocity is proportional to the square of
26
the particle size. Additionally, they have been praised for increased bioavailability of
27
several bioactive compounds (McClements, 2005; Sonneville-Aubrun, Simonnet, &
28
L’Alloret, 2004; Tadros, Izquierdo, Esquena, & Solans, 2004).
29
In our study, virgin olive oil was used to form an oil in water emulsion. This is
30
considered significant as many strategies and actions have been globally undertaken
31
by several initiatives and authorities to overcome obesity pandemic, known as
32
“globesity”. In particular, the World Health Organization suggestions towards fat
33
include a consumption shift from saturated to unsaturated fats and the restriction of
34
energy intake from fat (WHO, 2015).
35
Fenugreek (Trigonella foenum-graecum L.) seeds are generally used as a spice but
36
they also have medicinal properties. They contain 45% to 60% carbohydrate (mainly
37
galactomannan), 6% to 10% lipid (mainly polyunsaturated fatty acids), and 20% to
38
30% protein (4-hydroxyisoleucine being one of the major amino acids) (Rao,
39
Sesikeran, Rao, Naidu, Rao, & Ramacjandran, 1996; Raghuram, Sharma, &
40
Sivakumar, 1994). Over the last decade, the use of fenugreek gum has been increased
41
in many food products, because of its considerable thickening, stabilizing, and
42
emulsifying properties (Işıklı & Karababa, 2005). Additionally, fenugreek gum
43
contains a low amount of protein. These proteinaceous moieties, at concentration as
44
low as 0.1 wt %, impart an amphiphilic property to the gum, which can then act
45
somehow like an emulsifier (Brummer, Cui & Wang, 2003; Youssef, Wang, Cui, &
46
Barbut, 2009).
47
In a previous study (Kaltsa,Yanniotis & Mandala, 2016) we have shown that
48
fenugreek gum fractions with high galactomannan content (>80 %wt) may exhibit
49
similar or improved stability properties in whey protein emulsions compared to other
50
commercial galactomannans. However, when a high-energy homogenization process
51
is used, such as ultrasonication, a severe degradation of commonly used
52
polysaccharide stabilizers takes place, owed to extreme shearing. Hence,
53
polysaccharides’ addition in the initial formulation is not suggested, as their
54
degradation can reduce the viscosity of the continuous phase enhancing the
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ACCEPTED MANUSCRIPT destabilization process (Kaltsa, Michon, Yanniotis & Mandala, 2013; Kaltsa et al.,
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2016; Tiwari, Muthukumarappan, O'Donnell, & Cullen, 2010). Additionally,
57
stabilizers can hinder the efficacy of the homogenization process. They increase the
58
viscosity, which hinders the propagation of soundwaves, increases the cavitation
59
threshold to higher amplitudes and reduces the shearing turbulence (Wooster,
60
Golding, & Sanguansri, 2008).
61
The stability of whey prrotein nanoemulsions has been the topic of many studies
62
lately (Adjonu, Doran, Torley, & Agboola, 2014a, b; Ali, Mekhloufi, Huang, &
63
Agnely 2016; Ozturk, Argin, Ozilgen, & McClements, 2015; Teo et al., 2016; Zeeb,
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Herz, McClements, & Weiss, 2015). However, to our best of knowledge, there is no
65
available information regarding nanoemulsion models containing fenugreek gum.
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The objective of this study is the investigation of nanoemulsion formation by
67
changing the oil fraction, and ultrasonication processing time. In general low oil
68
fractions (<10 %) favor the formation of nanoemulsions below 200 nm, while higher
69
ones promote the formation of larger ones (Leong, Wooster, Kentish, & Ashokkumar,
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2009; Tabibiazar et al., 2015). Extended sonication time may also be useful in order to
71
reduce the mean droplet diameter of nanoemulsions as in a longer process there is
72
more time for the protein to orientate on the oil interface (Jafari Ηe, & Bhandari,
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2007). The stability of the final formulations (secondary model emulsions) produced
74
by further dilution with fenugreek gum was evaluated and comparisons were made
75
between nanoemulsions and their macroemulsion homologues produced by high-
76
speed mixing.
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2. Materials and methods
78
2.1. Materials
79
Whey protein isolate (WPI) Lacprodan DI-9224 was kindly provided by Arla (Arla
80
Foods Ingredients, Amba-Denmark). The composition of WPI powder in protein as
81
stated by the manufacturer was protein 92 ± 2 %, and maximum amounts of fat 0.2 %,
82
ash 4.5 % and lactose 0.2 %. Fenugreek gum powder Fenulife® was a kind gift from
83
Frutarom(Londerzeel, Belgium) (moisture content 6.3 %, soluble fiber content 83.2
84
%, mostly composed of galactomannan according to manufacturer and 3.2 % protein
85
calculated by Dumas method (Kaltsa, Yanniotis & Mandala, 2016)). Virgin olive oil
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Altis (Elais Unilever, Greece) was purchased from a local store. Sodium azide was
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from Sigma–Aldrich.
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2.2. Solution preparation
90
Whey protein stock solutions (10 g in deionized water in total weight 95, 90 or 80 g)
91
were prepared by agitation with a magnetic stirrer for 90 min. Fenugreek gum (FGF)
92
solutions 1, 1.5 and 2 %wt in deionized water were prepared by hot stirring in a water
93
bath at 90 oC for 90 min. Thereafter, solutions were kept overnight at 5 oC to ensure
94
complete hydration. Sodium azide 0.02 %wt was added to the aqueous solutions as an
95
antimicrobial agent.
96
2.3. Coarse emulsion preparation and screening
97
Oil-in-water primary macro-emulsions were prepared by homogenizing 5, 10 or 20 g
98
of oil phase with 95, 90 or 80 g of the WPI aqueous solutions accordingly. The initial
99
step in preparing macro-emulsions involved the production of a coarse premix
100
emulsion by homogenizing the oil and the aqueous phases using an Ultra Turrax (T25
101
basic, Janke & Kunkel IKA Labortechnik, Staufen, Germany) for 2 min (13.500
102
RPM) at room temperature. The coarse emulsions were further diluted with fenugreek
103
gum solutions (2 %wt) in a ratio of 1:1 by weight. Ingredients were further mixed
104
using a magnetic stirrer for 1 min in order to avoid recoalescence of the droplets and
105
to yield to the secondary coarse emulsions. Emulsion preparation was conducted in
106
neutral conditions in order to take advantage of the higher solubility of the protein.
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The pH of the resulting macro- and nano-emulsions was then adjusted to 3.8, which is
108
typical for acidic food emulsions, with a few drops of glacial acetic acid.
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Nanoemulsions were produced by further homogenizing 10 ml of the primary coarse
110
premixes with an ultrasonic homogenizer model Sonopuls 3200 (Bandelin Electronic
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Gmbh & Co, Berlin) equipped with a 3 mm in diameter titanium probe (MS 73). The
112
tip was immersed 10 mm from the surface of the sample which was placed in a double
113
jacketed flow-through vessel (model DG-3). The temperature was maintained at 30 ±
114
1oC by circulating cold water with a pump. The amplitude was set at 40%. It was
115
noticed that amplitude values higher than 40 % led to increased fluctuations of the
116
samples in the vessel, which caused sample spilling. The processing time ranged
117
between 1 up to 12 min. The experimental data at time intervals of 1, 2, 4, 8 and 12
118
min showed that the nominal ultrasonic energy input (kJ), as a function of time,
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The emulsification time, which resulted in both minimum droplet sizes and
121
polydispersity index (PDI) for all oil concentrations used, was chosen for the
122
incorporation of nanoemulsions in 2 %wt fenugreek solutions as mentioned above for
123
macro-emulsions. All samples were prepared in triplicate.
124
2.4. Droplet size measurement
125
Droplet size distributions of the primary coarse emulsions were determined using a
126
Mastersizer 2000 (Malvern Instruments, Malvern, UK). Emulsion droplets were
127
characterized under high dilution conditions by dispersing the samples in deionized
128
water. The refractive indices of water and olive oil were taken as 1.330 and 1.467,
129
respectively, and the Mie theory was used for the analysis (Paximada, Tsouko,
130
Kopsahelis, Koutinas, & Mandala, 2014). Average droplet sizes were characterized in
131
terms of the volume mean diameter D50 and polydispersity (Span). All measurements
132
were made at ambient temperature on three separately prepared samples.
133
The mean droplet size (z-average diameter) and the polydispersity index (PDI) of
134
nanoemulsions were measured using a dynamic light scattering instrument (Zetasizer
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Nano ZS, Malvern Instruments, Malvern, UK). Measurements were performed at
136
25 °C in triplicate. Prior to measurement, the nanoemulsions were diluted (1:500)
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with deionized water to avoid multiple scattering during measurements.
138
2.5. Emulsion stability
139
The storage stability (at 5 οC) of macro- and nanoemulsions was determined by
140
obtaining backscattering profiles with a Turbiscan 2000 device (Turbiscan 2000,
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Formulaction, Toulouse). For unstable macro emulsions the serum percentage was
142
calculated (Zinoviadou, Scholten, Moschakis, & Biliaderis, 2012). In stable samples
143
(macro- and nano/submicron emulsions), the average value of the backscattering (BS)
144
measurement at the middle zone of the cell (between 30 and 40 mm of sample height)
145
has been plotted as a function of ageing time and BS variation (dBS) during 10 days
146
of storage. Calculations were conducted as described in Kaltsa et al. (2013).
147
2.6. Emulsion rheology
148
Viscosity measurements were performed with a hybrid rheometer DHR (TA
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Instruments, New Castle, DE, US) equipped with plate-plate geometry (upper plate
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ACCEPTED MANUSCRIPT diameter 50 mm, gap 1 mm) to obtain flow curves between 0.1 to 1000 s-1 at 25 ±
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0.01 oC by circulating water from a peltier. The total measurement time was 900 s. All
152
measurements were performed in triplicate and data reported as average and standard
153
deviations.
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The linear viscoelastic region (LVR) was assessed at 6.28 rad/s (= 1 Hz) by strain
155
sweep experiments; for all samples a constant deformation of γ = 0.1 % was used,
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which was within the linear viscoelastic region of all the samples. Deviation from the
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linear viscoelastic region occurs when the sample starts to permanently deform,
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implying the destruction of the transient network structure. Small deformation
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oscillatory measurements were performed for the evaluation of the viscoelastic
160
properties, G′ (storage modulus), G″ (loss modulus), and tanδ (G″/G′) (Steffe,
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1996; Metzger, 2006), over the frequency range of 0.1–100 rad/s at 25 °C.
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3. Results and discussion
163
3.1. Droplet size
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The droplet mean diameter (D50) of primary coarse emulsions ranged between
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9.48 and 13.76 µm (Table 1). Although emulsion diameter usually increases when
166
increasing oil concentration (Christensen et al., 2001), only a slight change in the
167
mean diameter was observed for samples containing 20 %wt olive. Nevertheless, no
168
significant differences were observed compared to emulsions containing lower
169
amounts of olive oil (p>0.05). No significant differences were observed either
170
considering the polydispersity of samples since Span values ranged between 1.46 up
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to 2.48 (Table 1). This allows us to assume that the emulsification time and protein
172
isolate concentration used, were sufficient to equally minimize the droplet size in all
173
formulations examined regardless of the oil content.
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It is well known that WPI, as a protein, is a surface-active molecule that has the
175
tendency to adsorb at interfaces and undergoes structural rearrangement. However, its
176
solubility and emulsifying properties depend on pH. As mentioned above, emulsion
177
preparation was conducted at neutral conditions in order to take advantage of higher
178
solubility of the protein. In our study, the whey protein used, presented a minimum of
179
93.6 % solubility (data not shown) at pH 5, which is near the typical value of whey
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proteins’ pI ~ 4.5 (Pelegrine and Gasparetto, 2005) and should be mentioned that only
181
undenatured whey proteins remain soluble throughout all pΗ range
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ACCEPTED MANUSCRIPT Αt neutral pH values, oil droplets with whey protein absorbed at the interface possess
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a negative charge and are strongly repelled. On the contrary, at pH values near pI, the
184
surface charge diminishes and repulsive forces are weakened, leading to enhanced
185
attraction of the droplets. At even lower pH whey proteins take on a net positive
186
electrical charge. They can then interact with charge-carrying polysaccharides (e.g.
187
pectin, xanthan, carrageenan, chitosan) and extensive interactions may take place
188
either increasing or decreasing the stability of the emulsion. In our case, the reduction
189
of pH did not induce any instability, as fenugreek gum, like all other galactomannans,
190
does not carry any electrostatic charge to interact with the positively charged WPI on
191
droplet interface. However, proteinaceous moieties in fenugreek gums may impart an
192
amphiphilic property to the gum, which enables it to act somehow like an
193
emulsifier (Brummer et al., 2003; Youssef et al., 2009; Kaltsa et al., 2016).
194
Primary coarse emulsions were subjected to sonication in order to achieve further
195
droplet size reduction. As shown in Fig. 2a, a sharp decrease in the droplet size was
196
achieved within 1 min of sonication, hence the z-average diameter ranged between
197
323 - 288 nm depending on the oil content. Thereafter, the droplet size was
198
moderately reduced at increased sonication times up to 12 min. In more detail, the z-
199
average diameter of samples containing 5 and 10 %wt oil did not show any
200
considerable droplet reduction after 1 or 2 min of sonication accordingly, whereas a
201
longer time of 8 min was necessary that samples containing 20 % wt oil reach a
202
droplet size plateau.
203
Regarding the polydispersity of sonicated emulsions, it is shown that the PDI
204
decreased, when the sonication processing time increases (Fig. 2b). Although the PDI
205
values for samples containing 5 and 10 %wt oil, was 0.434 and 0.51 respectively after
206
1 min of sonication whereas the PDI values were considerably higher in samples
207
containing 20% olive oil. More specifically, emulsions containing 20 %wt olive oil
208
were characterized by
209
polydispersity of the droplets (Tiwari, Hihara, & Rawlins, 2014). However, results
210
should be treated with caution since DLS measurements can be considered reliable
211
when values of PDI <0.5 are obtained.
212
At low oil fractions 5 and 10 %, PDI values were unvarying after 4 or 8 min
213
accordingly. However, PDI values reduction under sonication time increase was
214
remarkable in samples containing 20% olive oil. Actually, the lowest PDI value was
215
observed at 12 min of sonication in emulsions containing 20% of olive oil.
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PDI values greater than 0.9, indicative of the strong
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ACCEPTED MANUSCRIPT Considering the above findings, a sonication time of 12 min was selected in order to
217
further add the FGF gum, as that time resulted in the lowest PDI values for all
218
samples produced.
219
Nanoemulsions with an average size droplets of 207 nm and a narrow polydispersity
220
(~ 0.2) at oil fraction 5% wt can be seen (Fig. 2a, b). This size is slightly higher than
221
that reported by Adjonu et al. (2014a), who prepared WPI nanoemulsions of 160 nm,
222
which could be attributed to slightly lower oil fraction used (4 %wt). Lower droplet
223
sizes reaching 75 nm have been referred by using bovine serum albumin (BSA) in the
224
presence of polyethylene-glycol (PEG) (Tabibiazar et al., 2015).
225
An increase in the oil fraction to 10 or 20% led to higher droplet sizes (~280 nm) and
226
PDI values (~0.38), although no significant differences were observed after 12 min of
227
sonication (p>0.05) (Table 1).
228
3.2. Screening of FGF concentration for secondary coarse emulsion preparation
229
Initially, a screening procedure was conducted in order to assess the FGF
230
concentration required to obtain stable secondary coarse emulsions containing 2.5, 5,
231
or 10 %wt olive oil in the final formulation. The curves of creaming (% Serum)
232
versus storage time of coarse emulsions containing 0.5 up to 1 %wt FGF are shown in
233
Fig. 3.
234
The oil phase content significantly affected the serum percentage values (p<0.05). As
235
can be seen the stability of emulsions was improved by increasing the oil content from
236
2.5 to 10 %wt (Fig. 3a, b,c).
237
For emulsions containing 0.5 %wt FGF the serum percentage decreased from 71.3 to
238
~ 9.5 % when the oil content changed from 2.5% to 10%wt, while for those
239
containing 0.75 %wt FGF, a reduction from 42.2 to 3.2 % was noticed for the same
240
oil content change. Stability improvement by increasing the oil content is well known.
241
It is associated to the increase in packing fraction of oil droplets, which leads to
242
enhanced interdroplet interactions and viscosity and to more structured emulsions
243
(Dickinson & Golding, 1997; Sun & Gunasekaran, 2009; Nikiforidis, Biliaderis, &
244
Kiosseoglou, 2012). However, the oil concentrations used are far from the droplet
245
close packing limits. In fact, creaming becomes inhibited altogether when the oil
246
volume fraction φ is as high that the droplets become close-packed, which is φ ~ 0.6
247
for monodisperse spheres (Dickinson, 2009).
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stability and prevent creaming through viscosity modification of the aqueous
250
continuous phase (Dickinson, 2003). In our case, the addition of 1 %wt FGF resulted
251
in the formation of stable coarse emulsions, which did not show any phase separation
252
during 10 days of storage. Therefore, this concentration was used in subsequent
253
formulations and comparisons were made between coarse and nanoemulsion
254
emulsions. Huang, Kakuda, & Cui (2001), who compared the stability of emulsions
255
containing different hydrocolloids, have also shown that long term stability without
256
phase separation could be achieved by the use of higher concentration of fenugreek,
257
guar and locust gums (1-1.5 %wt), attributed to a droplet movement restriction caused
258
by the continuous phase viscosity increase.
259
3.3. Emulsion rheological properties
260
The Power law model was adopted to describe the viscosity properties of coarse and
261
nanoemulsion analogues, as the same model was applied to describe the shear
262
thinning behavior of extruded fenugreek gum solutions (Chang & Cui, 2011).
263
Table 2 summarizes the estimated consistency (k) and flow behavior (n) values of
264
prepared secondary coarse and nanoemulsions containing 1 %wt FGF and various
265
amounts of olive oil. All model emulsions prepared were highly pseudoplastic with
266
flow coefficient (n) values varying from 0.29 to 0.35.
267
Consistency and flow behavior values were both influenced by the oil content and the
268
preparation method used, but the greatest changes were noticed at 10% oil. At 2.5% or
269
5% oil content differences among the samples were not significant in most cases
270
(Table 2).
271
Considering the coarse emulsions, an increase of oil content from 2.5 to 10 %wt
272
increased the consistency of the samples as well, from ~7.4 to 11.7 Pa-sn. This
273
change of the oil fraction causes an upward shift of the flow curves to higher
274
viscosities, attributed to the increased packing of the oil droplets. This leads to
275
enhanced droplet interactions, thus to more viscous emulsions (Sun & Gunasekaran,
276
2009). Significant differences were observed among all coarse emulsions regardless
277
of their oil content (p<0.05). Nevertheless, this was also observed in nano-emulsions,
278
for which, k values increased from 8.59 to 15.69 Pa-sn. The decrease of the droplet
279
size within the submicron scale resulted in an increase of the k values by 16 up to 34
280
% depending on oil concentration. However, it may be noticed that no significant
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ACCEPTED MANUSCRIPT differences are found between the viscosity values (k) of submicron emulsions
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containing 2.5 and 5 %wt olive oil (p>0.05).
283
The oil fraction increase also led to an enhancement of the pseudoplastic character in
284
both coarse and nano-emulsions, as evidenced by the decrease in flow behavior values
285
(n). Flow behavior values ranged between 0.333 – 0.352 and 0.288 – 0.324 for coarse
286
and nanoemulsions respectively. Similar values were reported for 1 %wt fenugreek
287
solutions by Wu et al. (2009), indicating the predominant role of fenugreek on
288
emulsion pseudoplasticity, as found in a previous study (Kaltsa et al., 2016).
289
Nanoemulsions were more pseudoplastic in all cases, regardless of the oil
290
concentration (p<0.05).
291
Dynamic oscillatory shear tests were used to characterize the viscoelastic properties of
292
coarse and nano-emulsions containing 1 %wt FGF. The storage (G’) and loss (G”)
293
modulii measured, are shown in Fig. 4(a-c). Loss tangent (tanδ) was also used to
294
assess the elastic or viscous character predominance in the samples as shown in the
295
same figure.
296
Overall, samples exhibited frequency-dependent storage and loss modulus values,
297
which increased during frequency ramp from 0.1 to 100 rad/s. In all emulsions, G”
298
was dominant over G’ in low frequency region, thereby showing a liquid like
299
behavior. This implies that the most of the input energy cannot be stored at this low
300
frequency region, thus it dissipates through viscous flow. At higher frequencies
301
(approximately > 1 rad/s) a cross-over occurs and a solid-like behavior is observed for
302
all emulsion preparations (G’>G”). Additionally, the crossover point of the modulii is
303
shifted at lower frequencies in the viscoelastic spectra of nanoemulsions.
304
Entanglements of macromolecular solutions can result in such behavior according to
305
Ross-Murphy (1984). Moreover, when G’ is higher than G’’ over most of the
306
frequency range investigated, the formation of a weak three-dimensional network of
307
ordered chain segments can be evident. The same behavior was observed in
308
nanoemulsions of higher oil content (Fig. 4c). However, as the tanδ values were
309
higher than 0.3 over the entire frequency range (Fig. 4), emulsions maintain a viscous
310
character (Mandala et al., 2004). Only for a tanδ > 0.1, a weak gel behavior is
311
ascribed, whereas a strong gel is evident at tanδ values less than 0.1 (Wang & Cui,
312
2005). These findings can be explained by the fact that galactomannans, even at low
313
concentrations, are capable of forming highly viscous solutions due to their high water
314
binding capacity. Galactomannans, such as fenugreek and guar gum, do not form gels
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ACCEPTED MANUSCRIPT on their own, whereas for locust bean gum, high concentrations and
subzero
316
temperatures (Dea et al., 1977) or a long storage are required to form gels
317
(Richardson, Clark, Russell, Aymard, & Norton, 1999). According to Wu, Cui, Eskin
318
& Goff (2009) fenugreek gum solutions with concentration ranging between 0.5 -2
319
%wt present a liquid like behavior at lower frequencies (G”>G’) and a solid-like at
320
higher ones. Moreover, in another study, the mechanical spectra of 1 %wt fenugreek
321
gum aqueous dispersions showed a change of behavior depending on the molecular
322
weight. Fenugreek gum fractions with higher molecular weight (Mw≥ 18.3x105
323
g/mol) showed a concentrated polymer solution pattern, exhibiting modulus crossover
324
(G’>G’’) at high angular frequencies, as in our case, whereas those of lower Mw
325
presented a dilute polymer solution pattern, as evidenced by modulii values (G’’>G’)
326
and absence of crossover (Funami et al., 2008).
327
The droplet packing caused by oil fraction increase and sonication resulted in
328
emulsion thickening as evidenced by the enhancement of G’ and G’’ values (Sun &
329
Gunasekaran, 2009).
330
At the same time, a decrease of loss tangent occurred, revealing the increase of the
331
solid like behavior. Minor differences in the storage, loss modulus and tanδ were
332
detected between formulations with low amounts of oil (2.5 and 5 %wt). Obviously,
333
findings of dynamic properties are in-line with those obtained in respect with the flow
334
behavior of the emulsions.
335
3.4. Stability of coarse and nano- emulsions containing 1 %wt FGF
336
Coarse and nanoemulsion formulations at 1 %wt FGF did not exhibit any phase
337
separation within the period tested (10 days, 5οC), as back-scattering values were
338
constant during storage (Fig. 5). According to Mie theory the back scattering intensity
339
increases by a decrease of the particle size of the droplets, when their diameter is
340
greater than 500 nm while the opposite applies for those of smaller size e.g.
341
nanoemulsions. It can be noticed that the initial BS intensity values are affected by oil
342
content and emulsification method. According to several studies BS intensity is a
343
value influenced by the size and density of the oil particles in emulsions. It is
344
enhanced by the increase of the oil droplet number and by decreasing their size or the
345
size of the flocs present (Palazolo, Sorgentini, & Wagner, 2004 and 2005). In our case
346
the increase of the oil fraction from 2.5 to 10 %wt increased the initial BS values of
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ACCEPTED MANUSCRIPT coarse emulsions from 40.3 to 61.6 % and those of nanoemulsions from 45.6 to 71.8
348
%.
349
Concerning storage time, in coarse emulsions containing 5 and 10 %wt oil, a
350
reduction of approximately ~4 % in BS (dBS) was noticed within 10 days of storage,
351
while it reached a value of 11.4 % decrease in samples containing 2.5 %wt oil (Fig.
352
5c). The BS change (dBS) was lower in nanoemulsions and ranged between ~ 1-1.9
353
%, hence an improvement in the stability in terms of the coalescence-flocculation was
354
achieved, attributed to droplet size reduction and thickening of the samples as
355
discussed above.
356
Normally, an increase in BS should have been noticed with storage time, in the case
357
of nanoemulsions, as a consequence of coalescence or flocculation phenomena
358
occurring during storage as previously observed in other studies (Porras, Solans,
359
González, & Gutiérrez, 2008; Wulff-Pérez, Torcello-Gómez, Gálvez-Ruíz, & Martín-
360
Rodríguez, 2009). In our case, the reason for a back scattering decrease could be
361
explained by the fact that nanoemulsions may have undergone flocculation during the
362
incorportation of FGF solution and subsequent pH adjustment from 6.5 to 3.8 passing
363
through the isoelectric point of proteins. The net negative charge that the oil droplets
364
posse at high pH values is suppressed by the decrease of pH values near the pI of the
365
protein (4.8), thus a few droplets of an average diameter 200 - 300 nm could easily
366
form flocs, the size of which could be detected by the Turbiscan wave length.
367
Considering a long storage of five months, as demonstrated in Fig. 6 and 7, coarse
368
emulsions prepared with 2.5 %wt oil were the least stable, since phase separation
369
(serum) occurred as evidenced by a sharp BS decrease near zero values at the bottom
370
of the tube (Fig. 6). Phase separation of that sample is also shown in Fig. 7. For the
371
rest of the samples, BS decreased gradually along
372
coalescence-flocculation phenomena indicated by higher BS values on day 0 (purple
373
line) and lower ones one day 150 (red line).
374
4. Conclusions
375
Low oil content model emulsions were developed and stabilized by FGF addition.
376
Stable coarse formulations were achieved upon the addition of 1 wt % FGF in all
377
cases of oil content (2.5, 5 or 10 %wt). Emulsion stability was further ameliorated by
378
preparing nano/submicron emulsions by ultrasonic homogenization. Nanoemulsions
379
with the smallest droplet diameter (~200 nm) and PDI (~0.2) were produced at the
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the sample tube due to
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ACCEPTED MANUSCRIPT lowest oil content after 12 min of sonication. Droplet size reduction in the
381
nano/submicron scale influenced the viscoelastic properties of the emulsions, as more
382
structured and viscous samples were obtained. All nano/submicron emulsions
383
exhibited higher stability during storage compared to coarse models due to a droplet
384
size reduction and their enhanced viscoelastic properties. Our results demonstrate that
385
the presence of nanosized droplets is mostly beneficial in the case of producing very
386
low oil content (2.5 % wt) model emulsions, as long term stability is still evident.
387
Acknowledgements
388
This research has been co-financed by the European Union (European Social Fund –
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ESF) and Greek national funds through the Operational Program “Education and
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Lifelong Learning” of the National Strategic Reference Framework (NSRF) –
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Research Funding Program: Heracleitus II. Investing in knowledge society through
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the European Social Fund.
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The authors would like to thank Frutarom Belgium N.V. for kindly donating
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FenuLife® extract.
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Figure 1. Volume weighted size distributions of primary emulsions as affected by oil
535
concentration.
536
Figure 2. Droplet size (a) and polydispersity index (PDI) (b) of emulsions as a
537
function of sonication time and oil concentration. Diamonds correspond to 5 %,
538
squares 10 % and triangles to 20 % oil.
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Figure 3. Stability of coarse emulsions containing as affected by oil (diamonds, 2.5
540
%wt, squares 5 %wt and triangles 10 %wt) and FGF concentration (open symbols: 0.5
541
%wt, closed symbols: 0.75 %wt and “x”, all other emulsions containing 1 %wt).
542
Different letters indicate significant differences among samples (p<0.05).
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Figure 4. Effect of oil content on storage (G’, closed symbols) and loss modulus (G”,
544
open symbols) of emulsions containing a) 2.5 %wt (diamonds), b) 5 %wt (squares)
545
and c) 10 %wt (triangles) olive oil. Black symbols indicate coarse emulsions and red
546
ones nanoemulsions. Black circles correspond to loss tangent (tanδ) of course
547
emulsions and red circles of nanoemulsions
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Figure 5. Stability of emulsions containing 2.5 (diamonds), 5 (squares) and 10 %wt
549
oil (triangles): a) Coarse emulsions, b) Nanoemulsions and c) Back scattering
550
variation (dBS) after 10 days of storage (5 oC).a-d Different letters indicate significant
551
differences among samples (p<0.05).
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Figure 6. Back scattering profiles of coarse and nano- emulsions containing 1 %wt
553
FGF during storage (5 oC).
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Figure 7. Stability of coarse and nano- emulsions containing 1 %wt FGF after 5
555
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Table 1. Droplet size (volume median diameter (D50) and z-average diameter) and
558
polydispersity (Span and PDI) of primary emulsions as affected by olive oil
559
concentration and emulsification method applied.
560
Table 2. Estimated consistency (k) and flow behavior (n) values of coarse and nano-
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Figure 1 7
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565
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4 6 8 10 Sonication time (min)
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ACCEPTED MANUSCRIPT 566
Figure 3 100
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G', G" (Pa)
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Figure 5 100
80
Back scattering (%)
60
40
60
12 Nanoemulsions 10
40
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6 8 Storage days
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Figure 6
Oil content (%wt)
10
80%
80%
60%
60%
60%
40%
40%
40%
20%
20%
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0mm
0%
50mm
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Back Scattering 100% 80% 60% 40% 20% 0% 0mm
150d
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Back Scattering 100%
152d
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Back Scattering
80%
80%
60%
60%
40%
40%
20%
20%
0mm
50mm
100%
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0% 150d
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Back Scattering 100%
80%
0%
Nano-emulsions
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ACCEPTED MANUSCRIPT Figure 7 2.5 % wt
5 % wt
10 % wt
Coarse emulsion Nanoemulsion
Coarse emulsion Nanoemulsion
Coarse emulsion Nanoemulsion
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Table 1 Oil concentration (%wt) 5
10
20
D50 (µm)
9.73a ± 3.3
9.48a ± 4.09
13.76a ± 3.96
Span (-)
1.81a ± 0.23
2.68a ± 1.08
1.46a ± 0.54
207.3a ± 1.0
288.7b ± 17.6
267.3b ± 8.5
0.219a ± 0.006
0.381b ± 0.021
0.386b ± 0.009
Nano- emulsions Z-average diameter (nm)
581
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Different superscripts per parameter indicate significant differences among samples (p<0.05).
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Table 2 k (Pa-sn)
n (-)
2.5
7.40a ±0.45
0.352d ±0.006
5
8.31b ± 0.32
0.343d ± 0.011
10
11.72c ±1.05
0.333c ±0.003
2.5
8.59b ±0.71
5
9.09b ±0.35
10
15.69d ±0.24
Oil content (%wt)
584
a-d
585
2
0.324bc ±0.006 0.320b ±0.001 0.288a ±0.012
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ACCEPTED MANUSCRIPT Highlights •
Low oil content macro and nanoemulsion models were prepared containing fenugreek gum
•
Droplet size decrease enhanced the rheological properties of nanoemulsions
•
Nanosized droplets are mostly appreciated for emulsions with the lowest oil
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