Microemulsions as Potential Carriers of Nisin ... - ACS Publications

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Microemulsions as potential carriers of nisin: effect of composition on the structure and efficacy Maria D Chatzidaki, Konstantinos Papadimitriou, Voula Alexandraki, Eirini Tsirvouli, Zena Chakim, Aghiad Ghazal, Kell Mortensen, Anan Yaghmur, Stefan Salentinig, Vassiliki Papadimitriou, Effie Tsakalidou, and Aristotelis Xenakis Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02923 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 11, 2016

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Microemulsions as potential carriers of nisin: effect of composition on the structure and efficacy Maria D. Chatzidaki1,2, Konstantinos Papadimitriou3, Voula Alexandraki3, Eirini Tsirvouli1, Zena Chakim1, Aghiad Ghazal4, Kell Mortensen,4, Anan Yaghmur5, Stefan Salentinig6, Vassiliki Papadimitriou1, Effie Tsakalidou3, Aristotelis Xenakis1,2*

1

-Institute of Biology Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens, Greece 2

3

-MTM, Faculty of Science and Engineering, Örebro University, Sweden

-Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens 4

5

6

- Niels Bohr Institute, University of Copenhagen, Denmark

- Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

- Laboratory for Biointerfaces, Department Materials meet Life, Empa. Swiss Federal Laboratories for Materials Science and Technology, St. Gallen, Switzerland

ACS Paragon Plus Environment

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ABSTRACT

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers, in order to assure its effectiveness as bio-preservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by Electrical Conductivity measurements, Dynamic Light Scattering (DLS), Small Angle X-Ray Scattering (SAXS) and Electron Paramagnetic Resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles’ size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased; whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the Well Diffusion Assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations but no significant difference was observed between the two used vegetable oils (ROO and SO).


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KEYWORDS: W/O microemulsion, bacteriosins, nisin, Electrical conductivity, DLS, EPR, SAXS, Well Diffusion Assay


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INTRODUCTION Soft self-assembled systems, such as fluid isotropic phases (L2), exhibiting specific physicochemical properties and offering novel technological characteristics, are among the emerging systems for both basic and applied research.1-4 The basic idea in this research area is to introduce novel soft self-assembled nanoobjects for the encapsulation, protection, and effective transfer

of

bioactive

compounds

for

different

applications

in

food,5-8

cosmetics,9

pharmaceuticals,10-12 agrochemicals,13 and other industries while new applications are continuously being reported. Olive and sunflower oils are constituted of monounsaturated and polyunsaturated fats with low saturated fat levels. Olive oil is richer in oleic acid, whereas sunflower oil has a higher concentration of linoleic acid.14 In general, both oils are obtained by refining methods and thus are deprived from their minor components.15 Some of these minor components, such as phospholipids, phenols, and proteins have amphiphilic properties and may interfere with the used surfactant molecules in the reverse micelle structure leading to the formation of mixed micelles.16 Ethanol is a polar component used in such systems because of its characteristic ability to destabilize liquid crystalline phases thus enlarging the fluid isotropic monophasic micellar area.17 On the other hand, polar lipids such as monoglycerides are able to form a reversed micellar solution (L2 phase) when solubilized into triglycerides in the presence of water, a system that could be of physiological significance because it is one of the phases formed during fat digestion.18 In addition, monoglycerides are also widely investigated due to their ability to form complex liquid crystalline phases on exposure to water. More specifically for the latter case, the monoolein (MO) - water and monolinolein (MLO) -water systems have attracted significant attention in the literature owing to their ability to modulate self-assembled nanostructures


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including different inverse liquid crystalline phases and L2 phases by adding a suitable third component .17, 19-22 Bacteriocins, which are ribosomally synthesized proteinaceous compounds, usually kill bacteria phylogenetically close to the producer strain, though sometimes exhibit a broad host range activity, inhibiting many different species.23 Among these bioactive compounds, nisin is a well-known FDA-approved GRAS bacteriocin produced by Lactic Acid Bacteria (LAB) used in the food industry as a natural bio-preservative (E234) against food borne pathogens.24-26 However, there are numerous studies reporting on the proteolytic degradation of nisin and the loss of its effectiveness due to possible interactions with food ingredients.27-28 Therefore, much attention has been paid to assure the retaining of its effectiveness when administrated. In particular, nisin-loaded vehicles such as liposomes or solid lipid nanoparticles (SLNs) were proposed for optimizing bacteriocins’ antimicrobial activity and for enhancing its stability and efficacy against targeted bacterial species.29-30 However, these conventional methods generally require applying an external high-energy input in order to form the proposed structures while progressive phase separation may eventually occur. Since various peptides are amphiphilic molecules known for their ability to be adsorbed at fluid interfaces,31-32 here we examined the alterations on membrane dynamics and nisin’s effectiveness when encapsulated to the proposed inverted type lipid-based systems. The aim of the present study was to get insight into the formulation of inverse microemulsions (L2 phases) based on distilled monoglycerides (DMG) and refined olive oil (ROO) or sunflower oil (SO), as carriers of nisin. Structural characterization of the proposed soft self-assembled nanosystems was obtained using electrical conductivity measurements, Dynamic Light Scattering (DLS), small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) spectroscopy in order to investigate the effect of increasing ethanol concentration, as well as the


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nature of the used vegetable oils, on the structural characteristics of the formed inverse microemulsions. The effect of loading nisin on the reverse micelles was also determined. Furthermore, an in vitro assay was introduced in order to assess the potential efficacy of nisinencapsulated inverse micellar nanoobjects against L. lactis as related to their structural characteristics.

EXPERIMENTAL SECTION Materials. Refined olive oil (ROO) was kindly provided by ELAIS S.A.-Unilever (Greece). Sunflower oil (SO) was a commercial product purchased from a local market. Distilled Monoglycerides (DMG) containing monolinolein (C 18:2) up to 74 %, were kindly provided by Palsgaard (Denmark). Ethanol was from Merck (Darmstadt, Germany). Nisin N5764 and 5-doxyl stearic acid (5-DSA) were purchased from Sigma-Aldrich (Germany). Μ17 broth medium was purchased from Oxoid Ltd., Basingstoke, Hampshire (UK) and D(+)-Glucose was from AppliChem, Darmstadt (Germany). Ultra-pure water was obtained from a Millipore Milli Q Plus device. Microemulsion Formulation. Mixtures of DMG: oil (binary mixture of vegetable oil: ethanol) were prepared under the constant ratio 40 emulsifier: 60% w/w oil for all experiments. Water or nisin stock aqueous solution (nisin dissolved in water according to the protocol that follows) was added dropwise at specific concentrations, depending on the experiment, to obtain a transparent micellar solution that still remains in the monophasic region (detailed phase diagram is provided in the Supporting Information, Fig.S1). Vegetable oil (ROO or SO): ethanol binary mixtures were prepared at the following ethanol mass fraction (Wethanol): 0.00, 0.08, 0.17, 0.25, 0.33, 0.40 and 0.50.


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Nisin solution was prepared as follows: 40 mg of nisin (2.5% nisin with sodium chloride and denatured milk solids as balance) were dissolved in 1 mL of 0.05% (v/v) acetic acid and the pH was adjusted at 5.5. After 10 min at room temperature, the insoluble material was removed by centrifugation (10 min, 13.000 rpm, 25ºC). Nisin remained completely dissolved in the clear supernatant (2.560 AU/mL) and was used for the encapsulation experiments. For SAXS experiments, the effect of ethanol was investigated on inverse DMG/ROO- and DMG/SO-based microemulsion at a constant 40:60 weight ratio (DMG: oil (binary vegetable oil - ethanol mixture)) containing 4 and 2% water, respectively. Different ethanol mass fractions of the vegetable oil - ethanol mixture (Wethanol= 0.00, 0.25, 0.33, 0.40, 0.50) were examined. The hydration effect was evaluated at a constant 40:60 weight ratio (DMG: oil (binary mixture of vegetable oil: ethanol)) with the addition of 4, 6, 9% and 2, 5, 6% water for DMG/ROO- and DMG/SO-based systems, respectively. Wethanol was kept constant at 0.50. Electrical Conductivity. The electrical conductivity was measured with a Metrohm 644 conductometer at 25±1 °C using a thermostated microcell. The cell constant, c, was equal to 0.1 cm-1. Empty W/O microemulsions were constructed as described above. Conductivity was measured for increasing water content. More specifically, mixtures of surfactant: oil weight ratio 40/60 (DMG/ oil (binary mixture ROO- ethanol)) were prepared. Ethanol mass fraction of the ROO: ethanol mixture was kept constant at 0.50. Then, water containing 2 mM NaCl was added to obtain systems containing 0, 4, 6, and 9 wt. % aqueous phase. Dynamic Light Scattering measurements. DLS measurements were performed using an ALV/CGS-3 Compact Goniometer System (ALV GmbH, Germany) coupled JDS Uniphase 22mW equipped with a He–Ne laser (λ=632.8 nm). The average hydrodynamic radius of the structures was measured at detections angles of 130° or 150°. The temperature was 25±0.1°C.


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ROO was considered as the solvent with a viscosity of 67.0 mPas (cf. Supporting Informationviscosity measurements). The Refractive Index of ROO was measured using a Kern ORT 1RS Abbe refractometer and was found 1.47. Aqueous and oily phases were filtered through 0.45 µm filters before measurements. Autocorrelation functions were collected for each sample, and were analyzed by the cumulant expansion method using the CONTIN routine. The hydrodynamic radii were calculated using the Stokes-Einstein equation for diffusion coefficients and the resulted distribution functions were weighted per intensity. The water addition was evaluated at a constant 40:60 weight ratio (DMG: oil (binary mixture of ROO: ethanol)) with the addition of 5 and 9 % of water. Mass fraction of ethanol was kept constant at 0.50. Samples without water were also measured. Small-Angle X-ray Scattering (SAXS) measurements. SAXS was performed using the GANESHA-SAXS/WAXS apparatus (SAXSLAB, Denmark). Measurements were performed using Cu-Kα X-ray radiation in a q range of around 0.05-0.8Å-1, q being the magnitude of the scattering vector q=4π/λsin(θ), where λ = 1.54 Å is the X-ray wavelength and θ is half of the scattering angle. The scattering profiles of inverted-type water-in-oil (W/O) microemulsions (L2 phases) show a single broad peak. The d spacing (characteristic distance) for these phases was calculated for each scattered curve using the relationship d =

∗

, where q*are the scattering broad

peaks obtained from the X-ray spectra. The empty capillary was measured and subtracted as background scatter from all experimental data. The sample holders used were 2mm thick capillaries loaded in a controlled temperature chamber from Linkman, set to 25 and 37°C. The software used was SPEC (CSS, Certified Scientific Software, Cambridge, USA) to control the instrument and SAXSGUI (SAXSLAB, Denmark) to make the basic data evaluation.


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X-ray data analysis. It should be noted that inverse micellar solutions consist of small droplets in a continuous matrix or as bicontinuous phase where a monolayer of surfactant film separates oil and water domains. The Teubner Strey model, developed for bicontinuous phases, did not provide a reasonable fit to our SAXS data. This indicates that the system is in the nanoparticulate regime33. Hence, we assume microemulsion droplets with a hydrophilic core and a hydrophobic shell.34-35 In case of monodisperse, homogeneous and spherical particles, the SAXS scattering intensity can be expressed as following: I(q)=NS(q)P(q)

(1)

where, N is the number of particles, S(q) is the structure factor describing inter-particle interactions, and P(q) is the form factor describing intra-particle interactions.36 The form factor of spherical core-shell micelles was calculated using: =

3 −

!

()*+,-./01

+ 3 − #$%&

!

' +

(2)

where scale is a scale factor, V is the volume, r is the radius, ρ the electron density with the indices s for shell and c for the core.36 This model provides characteristic parameters such as core- and shell dimensions and relative differences in electron densities between shell and core. The value for scale was fixed at 0.4, corresponding to the mass fraction of the used emulsifier, and ρsolvent was estimated to be 9.02*10-6 Å-2, the electron density of triolein. The values for r and ρ for core and shell were optimized to achieve the best possible fit to the SAXS data. The polydispersity was included by calculating a Gaussian distribution of polydisperse particles that


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was fixed at 5% for σ/rc and σ/rs. It is worth mentioning that the scattering of non-spherical droplets can also be described with a model of polydisperse spheres. The effective inter-particle (structure) factor for spherical droplets interacting through hard sphere interactions was evaluated using the Percus-Yevick approximation.37 It should be noted that SAXS was used to investigate the inverse micellar solutions for shedding light on the detected structural effects in terms of hydration and ethanol concentration at constant compositions of the emulsifier and oil phase (a binary vegetable oil – ethanol mixture). Also, the systems were investigated for possible alterations in the structure when changing the nature of the used vegetable oil. EPR measurements. EPR spectra were recorded at approximately 25°C, using a Bruker EMX EPR spectrometer operating at the X-Band. Samples were incubated in a WG-813-Q Wilmad (Buena, NJ) Suprasil flat cell. The amphiphilic 5-doxyl stearic acid (5-DSA) spin probe was used to study the interfacial properties of W/O microemulsions. Stock solutions of 10-2 M of the spin probe in ethanol were prepared. After ethanol evaporation, 1 mL of each microemulsion was added in order to obtain a final concentration of 10-4 M 5-DSA in the microemulsion. Results were analyzed in terms of rotational correlation time (τR) and order parameter (S).38-39 Calculation of the rotational correlation time (τR) The rotational correlation time, τR, of the spin probe was calculated from the EPR spectra using the following relationship: 23 = 6 × 10

89

ℎ9 8/ ℎ9 8/ :; > +; > − 2A BC9 , E 3

ℎ=8 ℎ8


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∆Η0 is the width of the central peak and h+1, h0, and h-1 are the intensities of the low, center and high field peaks of the EPR spectrum, respectively. The relationship in Eq. 3 is applicable in the fast motion region, i.e. for correlation times in the range of 10-11

Microemulsions as Potential Carriers of Nisin ... - ACS Publications

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