Influence of spray drying on the stability of food-grade solid lipid nanoparticles

Accepted Manuscript Influence of spray drying on the stability of food-grade solid lipid nanoparticles

Hanna Salminen, Juliane Ankenbrand, Benjamin Zeeb, Gabriela Badolato Böhnish, Christian Schäfer, Reinhard Kohlus, Jochen Weiss PII: DOI: Reference:

S0963-9969(18)30848-2 doi:10.1016/j.foodres.2018.10.056 FRIN 8026

To appear in:

Food Research International

Received date: Revised date: Accepted date:

22 June 2018 9 October 2018 21 October 2018

Please cite this article as: Hanna Salminen, Juliane Ankenbrand, Benjamin Zeeb, Gabriela Badolato Böhnish, Christian Schäfer, Reinhard Kohlus, Jochen Weiss , Influence of spray drying on the stability of food-grade solid lipid nanoparticles. Frin (2018), doi:10.1016/ j.foodres.2018.10.056

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ACCEPTED MANUSCRIPT Influence of spray drying on the stability of food-grade solid lipid nanoparticles

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Hanna Salminen*, Juliane Ankenbrand, Benjamin Zeeb, Gabriela Badolato Böhnish, Christian Schäfer, Reinhard Kohlus, Jochen Weiss

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Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, 70599 Stuttgart, Germany

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DSM Nutritional Products Ltd., Research Center Formulation & Application, P.O. Box 2676, 4002 Basel, Switzerland

Submitted to Food Research International, June 2018

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Revised October 2018

KEYWORDS:

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Spray drying; Solid lipid nanoparticles; Nanostructured lipid carriers; -3 fatty acids; Quillaja saponins; Polymorphic transition; Maltodextrin

*Corresponding author: H. Salminen E-mail address: hanna.salminen@uni- hohenheim.de Tel. +49 711 459-24457; Fax: +49 711 459-24446

ACCEPTED MANUSCRIPT ABSTRACT This study investigated spray drying of food-grade solid lipid particles (SLN) and nanostructured lipid carriers (NLC) containing -3 fish oil. Stable SLN and NLC dispersions with tristearin as carrier lipid were formed by using a combination of Quillaja saponins and high-melting lecithin

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as emulsifiers. Our specific goal was to study the influence of four different spray drying inlet

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and outlet temperatures (Tinlet/outlet = 140-170C/65-95C) and two different maltodextrin types

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(DE 6 and DE 21) with different molecular weights as protective wall materials on the physical

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and polymorphic stability of the solid lipid particles. The results revealed that the low molecular weight maltodextrin DE 21 was a superior wall material for stabilizing the solid lipid particles.

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Moreover, the lipid particles spray dried at Tinlet/outlet of 140/65C exhibited the highest physical and polymorphic stability, whereas using higher Tinlet/outlet led to bigger particles which were

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more prone to polymorphic transition. This was also verified in a 71-day storage test. The

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findings were explained that by preventing the melting of the tristearin carrier lipid during spray

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drying, the crystallized lipid particles remained intact inside the amorphous maltodextrin layer and exhibit high physical and polymorphic stability. These findings are important for generating

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stable food-grade spray dried powders.

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ACCEPTED MANUSCRIPT 1.

INTRODUCTION

Spray drying is a widely used method for improving shelf-life of foods and food ingredients since dehydration during the process results in a low water content that in turn will reduce chemical, physical and biological degradation of the product. The conversion of a liquid material

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into a dry powder offers multiple advantages for industrial use: concentration and encapsulation

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of bioactive ingredients, extended storage stability due to less molecular mobility, and reduced

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transportation and storage costs (Baker, 1997; Gharsallaoui, Roudaut, Chambin, Voilley, &

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Saurel, 2007).

Encapsulation matrices such as solid lipid nanoparticles (SLN) and nanostructured lipid

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carriers (NLC) have been shown to be efficient in protecting bioactive ingredients against

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chemical degradation (Helgason, Awad, Kristbergsson, Decker, McClements et al., 2009; Salminen, Aulbach, Leuenberger, Tedeschi, & Weiss, 2014a; Salminen, Gömmel, Leuenberger,

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& Weiss, 2016; Salminen, Helgason, Kristinsson, Kristbergsson, & Weiss, 2013). SLN are fully

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crystalline lipid matrixes in which other crystalline and hydrophobic actives can be incorporated.

liquid lipids.

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NLC, on the other hand, have a partially crystalline structure composed of a mixture of solid and

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Although spray drying has been applied frequently for other types of colloidal systems (Baker, 1997; Drusch, Serfert, van Den Heuvel, & Schwarz, 2006; Gharsallaoui, et al., 2007; Hernandez Sanchez, Cuvelier, & Turchiuli, 2015; Huang, Hao, Li, Yang, Cen et al., 2014), its use for solid lipid particles has been rather limited. The literature on spray drying of SLN or NLC

reports

that the formulations (types of lipids,

emulsifiers,

and

wall materials),

homogenization and spray drying techniques used varied considerably (Freitas, & Müller, 1998; Gaspar, Serra, Lino, Gonçalves, Taboada et al., 2017; Wang, Hu, Zhou, Xia, Nieh et al., 2016a; 2

ACCEPTED MANUSCRIPT Wang, Hu, Zhou, Xue, & Luo, 2016b; Wang, Ma, Lei, & Luo, 2016). Some of the first spray drying studies of SLN in the pharmaceutical field were already conducted in the 1990’s, however, this was shown to be very challenging (Freitas, & Müller, 1998): The authors showed that spray dried SLN (glycerol behenate as lipid phase, polaxamer 188 as emulsifier) could be

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redispersed to similar sizes as the original SLN before spray drying (for intravenous therapy

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purposes) only when using very low lipid concentrations (1%) and applying trehalose as spray

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drying matrix. Some recent papers also report spray drying of SLN, however, this has required the use of additional stabilization methods such as covering the SLN (glyceryl dibehenate

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emulsified with Tween 80) with an additional protein layer followed by spray drying with

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trehalose or mannitol (Gaspar, et al., 2017). Other studies have described the spray drying of SLN (glyceryl dibehenate emulsified with sodium caseinate) covered with a layer of pectin

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followed by chemical crosslinking (Wang, Ma, Lei, & Luo, 2016) or spray drying of SLN

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(glycerol dibehenate emulsified with lecithin) covered with two additional biopolymer layers (Na-caseinate and pectin) via layer-by-layer deposition (Wang, et al., 2016a). Another study

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showed that spray drying of SLN with different polysaccharides such as pectin, alginate,

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carrageenan, carboxymethyl cellulose, or gum arabic as wall materials was unsuccessful and led to formation of aggregated powders (Wang, et al., 2016b). The authors, however, reported that

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spray drying of NLC containing 20-30% oleic acid with carrageenan or pectin as protective agent formed small, spherical powder particles (Wang, et al., 2016b). However, none of these studies investigated the physical and polymorphic stability of the solid lipid particles after spray drying (or even before spray drying) over time, which is crucial when the solid lipid matrix is prone to polymorphic transition. Furthermore, all these studies demonstrate that the underlying instability phenomenon occurring in crystallized lipid particles due to recrystallization behavior was not

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ACCEPTED MANUSCRIPT solved (nor even investigated), as these studies used polysorbates, lecithins, or polaxamers as primary emulsifiers (Freitas, & Müller, 1998; Gaspar, et al., 2017; Wang, et al., 2016a; Wang, et al., 2016b; Wang, Ma, Lei, & Luo, 2016) which are not able to physically stabilize solid lipid particles as has been revealed in earlier studies (Helgason, Awad, Kristbergsson, McClements, &

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Weiss, 2008; Salminen, Helgason, Aulbach, Kristinsson, Kristbergsson et al., 2014b).

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Therefore, our approach was to spray dry SLN and NLC that are physically stable prior spray

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drying. The formation of such physically stable – and food-grade – solid lipid particles has been

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described in our earlier studies (Salminen, et al., 2014a; Salminen, Gömmel, Leuenberger, & Weiss, 2016): For the stabilization of the solid lipid particles, a combination of Quillaja saponin

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extract and high-melting lecithin was used as these emulsifiers can inhibit polymorphic transition

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of the crystallized carrier lipid, and form physically stable lipid particles, which will then ensure a homogeneous matrix for spray drying. Both Quillaja saponin extract and lecithins are approved

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for food use (Commission Regulation (EU), 2011; US Food and Drug Administration, 2017). We

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postulated that by using appropriate spray drying parameters and applying a suitable wall material we can ensure the stability of the solid lipid particles upon spray drying as long as we

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can prevent the melting of the carrier lipid during spray drying. To achieve this, Freitas & Müller

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(1998) recommended the use of lipids with a melting point  65C and addition of a carbohydrate as protective wall material. For this purpose, we investigated the impact of spray drying on the physical and polymorphic stability of SLN and NLC containing -3 fish oil, which has been shown to be essential for good brain and cardiovascular health (Colussi, Catena, Novello, Bertin, & Sechi, 2017; Dyall, 2015). In particular, we evaluated the impact of spray drying inlet temperatures (140-170ºC) as well as two different wall materials to prevent destabilization of the solid lipid particles. As wall materials, we used either low or high 4

ACCEPTED MANUSCRIPT molecular weight maltodextrins, which are commonly used in spray drying (Alamilla-Beltrán, Chanona-Pérez, Jiménez-Aparicio, & Gutiérrez-López, 2005; Descamps, Palzer, Roos, & Fitzpatrick, 2013; Gharsallaoui, et al., 2007). Freeze drying was used as a reference process as it

MATERIALS AND METHODS

2.1

Materials

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2.

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is usually applied for sensitive ingredients.

MEG-3TM ´30´n-3 Food Oil (DSM Nutritional Products Ltd., Basel, Switzerland) consists of

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refined fish oils with ≥ 30% ω-3 polyunsaturated fatty acids as triglycerides (eicosapentaenoic acid ≥9%, docosahexaenoic acid ≥12.5%). Tristearin (Dynasan 118) containing ≥ 97 % stearic

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acid with a melting point ~73 °C was a donated by Cremer Oleo GmbH & Co. KG (Witten,

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Germany). Quillaja extract (Andean QDP Ultra Organic, Desert King Int., San Diego, CA, USA), composed of > 60.0% saponins and < 3.38% citric acid, was purchased from PERA

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GmbH (Springe-Eldagsen, Germany). Phospholipon 80H, a high melting lecithin containing ≥

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70% hydrogenated phosphatidylcholine and ≤ 6% hydrogenated lysophosphatidylcholine from soybean was a gift from Lipoid GmbH (Ludwigshafen, Germany). Maltodextrin DE 6 and DE 21

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(DE = dextrose equivalent) were both amorphous spray dried powders obtained through

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enzymatic hydrolysis from maize starch containing mixtures of α-1,4 linked oligosaccharides with occasional α-1,6 branches and were purchased from Rouquette GmbH (Frankfurt, Germany). Maltodextrin DE 6 (Glucidex 6, MW ~ 93 kDA) contained ~99% higher polysaccharides, ~1% disaccharides and ~0.5% glucose, and maltodextrin DE 21 (Glucidex 21, MW ~ 7.2 kDa) ~92% higher polysaccharides, ~7% disaccharides and ~1% glucose. Sodium phosphate monobasic (purity ≥ 99%), sodium phosphate dibasic (purity ≥ 99%), and sodium

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ACCEPTED MANUSCRIPT azide (purity ≥ 99%), were obtained from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Bidistilled, deionized water was used for all experiments. 2.2

Preparation of lipid nanoparticles

A surfactant solution (3% w/w) was prepared by dissolving 2.4% (w/w) Quillaja extract and

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0.6% (w/w) lecithin in 10 mM sodium phosphate buffer (pH 7) containing sodium azide (0.02%

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w/w) as bacteriostatic agent, stirred for 20 min and then heated up to 85°C for 10 min. Lipid nanoparticles (SLN and NLC) with 10% (w/w) lipid phase were generated using the

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hot high-pressure homogenization method. For SLN, tristearin (10% w/w) was fully melted at 85°C and then added into the surfactant solution (90% w/w). For NLC, the melted tristearin (8%

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w/w) was mixed with heated (85°C, 5 min) ω-3 fish oil and then added into the surfactant

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solution (90% w/w). A pre-emulsion was then formed by using an ultra-turrax (IKA, Staufen, Germany) at 24000 rpm for 2 min. A hot high-pressure homogenization process was then carried

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out in a Microfluidizer Processor M-110EH (Microfluidics Corporation, MA, USA) equipped

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with a H10Z interaction chamber (diameter: 100 μm) at 500 bar in four cycles. In order to prevent the crystallization, hot water was cycled through the homogenizer, and the sample was

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kept at 85°C between each cycle. The final emulsion was cooled down in an ice bath for 1 h to

2.3

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induce crystallization. Spray drying

Prior spray drying, 20% (w/w) of maltodextrin powder (DE 21 or DE 6) was added directly into the emulsion and stirred overnight (12-15 h) to ensure complete dissolution and homogeneity. A mini spray dryer (B-290, Büchi, Switzerland) operating in a co-current flow was used to spray dry the lipid nanoparticles. The co-current flow can be an advantage for heat sensitive products as the hottest air is only in contact with the sample right after its atomization and not with the dry 6

ACCEPTED MANUSCRIPT powder. The samples were dried at a feed rate of 6 cm³/min and an atomizing air pressure of 5 bar. The aspirator suction was set at 633 dm³/min. With the used nozzle (d=0.7 mm) a droplet size between 1 to 25 μm could be achieved according to the manufacturer. The mean residence times of the particles were between 1 and 1.5 s. Tinlet were varied from 140°C to 170°C which led

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to Toutlet between 65°C and 95°C (Table 1). The generated powder was stored in a desiccator.

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The powder yield (in %) was calculated as follows: (total weight of powder / total weight of

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solids added initially) x 100, where the total weight of solids is based on the sum of the initial

2.4

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solids (lipids, surfactants, and maltodextrin) and the residual water content. Freeze drying

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The lipid particles were dried by using a freeze drying unit Type L-10 (WKF Forschungsgeräte

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GmbH, Darmstadt, Germany). The samples (50 g each) were placed in aluminum bowls and placed in the freeze dryer. First, the samples were frozen to -40°C within 4 h and stored at that

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temperature for 2 h. A vacuum (50 Pa) was created, and the samples were dried for 24 h. The dry

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cake was then ground to get a free-flowing powder. Moisture content and aw of powders

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The moisture content of the powders was determined by using a Karl Fischer titration setup (803

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Ti Stand, Metrohm, Switzerland) equipped with a titrator (Titrando 841) and a 20-mL burette. For the measurement, a two-component system was used with a hydranal-solvent and a hydranaltitrant (both Sigma-Aldrich, Germany). The water activity (aw) of the powder was determined at 25°C using an aw Sprint TH-500 water activity meter (Novasina, Switzerland). 2.6

Particle size

Particle sizes were determined using a static light scattering device (Horiba LA-950, Retsch Technology GmbH, Haan, Germany). Refractive indexes of 1.54 and 1.33 were used for the lipid 7

ACCEPTED MANUSCRIPT particles and dispersion medium, respectively. The spray dried powders were redispersed in 10 mM sodium phosphate buffer (pH 7) (1:10) overnight before measurements. Redispersing the spray dried powder to aqueous medium results in release of the solid lipid particles from the powder; therefore, this method does not measure the powder particle size but only the size of the

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SLN and NLC after redispersion. All samples were further diluted to approximately 0.02% in the

i=1

nidi3 , where ni is the number of particles with a geometric diameter

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diameters d43 =  i=1 nidi4 /

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buffer to avoid multiple scattering effects. The results are shown as mean volume-based particle

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di. Scanning electron microscopy (SEM)

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Dried powders were placed on an aluminum stub and were sputter coated (SCD 040, Optics

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Balzers AG, Balzers, Liechtenstein) with a mixture of gold-palladium steam (20:80) for 8 min. The samples were examined using a Zeiss DSM 940 scanning electron microscope (Carl Zeiss

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AG, Oberkochen, Germany). The working distance was set at 6 mm and an accelerating voltage

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of the electron beam of 5000 V was used. Images at a magnification of 1000x were taken of powders at day 1 and day 71 of the storage period. Differential scanning calorimetry (DSC)

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2.8

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Differential scanning calorimeter DSC 8500 (Perkin Elmer Inc. Shelton, CT, USA) was used to identify the crystal structure and the thermal behavior of the samples. The spray dried powders were redispersed in 10 mM sodium phosphate buffer (pH 7) (1:10) overnight before measurements. An aliquot of ~10 mg of sample was placed into an aluminum pan and hermetically sealed with an aluminum lid. The sample was heated from 20°C to 85°C and then cooled down to 10°C at 20°C/min. An empty sealed pan was used as a reference. 2.9

Storage test 8

ACCEPTED MANUSCRIPT Storage tests over 71 days were carried out for selected samples as shown in Table 2. All samples contained 20% (w/w) maltodextrin DE 21. Maltodextrin DE 6 was not used for the storage tests. The powders (10 g) were filled into polyamide/polyethylene plastic bags (170 x 220 mm, 90 μm) right after spray/freeze drying, and the headspace air was removed before (Kammermaschine

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400,

Multivac Sepp

Haggenmüller GmbH & Co.

KG,

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sealing

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Wolfertschwenden, Germany). The liquid samples were stored in closed glass containers. All the

Sensory analysis

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2.10

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samples were kept in the dark at the above specified storage temperatures (4 or 35°C).

The spray and freeze dried powders selected for the storage test (section 2.9) were subjected to

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sensory analysis. Both spray and freeze dried NLC powders contained the same fish oil

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concentrations (Table A.1). The SLN powders contained no fish oil (Table A.1), and therefore served as negative references in the sensory analysis. The powders were stored in the dark for 5,

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19, 41 and 68 days. At least 10 panelists classified the powders into five sensory attributes -

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fishy, green, mouldy, sweet and cooked – on an intensity scale from 1 (low intensity) to 7 (strong intensity) by sniffing. If an attribute could not be smelled no value was given. These attributes

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were based on a previous sensory investigation of fish oil powders (Serfert, Drusch, Schmidt-

samples. 2.11

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Hansberg, Kind, & Schwarz, 2009). Coffee beans served as neutralization smell between

Statistical analysis

All experiments were repeated at least two times using freshly prepared samples. Means and standard deviations were calculated from a minimum of three measurements using Excel (Microsoft, Redmond, WA, USA). The data was analyzed by means of ANOVA (p0.05) (Sigma Plot 12.5, Systat Software Inc., Chicago, USA). 9

ACCEPTED MANUSCRIPT 3.

RESULTS AND DISCUSSION

3.1

Characterization of spray dried lipid particles

The purpose of these experiments was to evaluate the impact of Tinlet/outlet and maltodextrin type on the physical stability and thermal behavior of SLN and NLC upon spray drying. The exact

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ingredient composition of all the samples in solution and dry state can be found in

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3.1.1

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Supplementary material (Table A.1). Yield

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The yields of the spray dried powders varied depending on the type of maltodextrin used:

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Maltodextrin DE 21 showed yields around 60-66%, whereas maltodextrin DE 6 resulted only in 20-33% powder yields (Table 1). These unexpectedly low yields with maltodextrin DE 6 can be

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explained mostly by viscosity effects as maltodextrin DE 6 has a higher molecular weight (93

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kDa) and thus a higher viscosity than the lower molecular weight maltodextrin DE 21 (7.2 kDa) (Descamps, Palzer, & Zuercher, 2009; Serfert, et al., 2009). The laboratory scale spray tower

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used for this investigation has a very short path from the atomizer to the spray tower wall.

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Therefore, the yield from this type of spray tower is mostly influenced by how fast the droplets dry before they hit the spray tower wall. When the viscosity of the feed increases, the droplet

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size will increase during atomization (Ré, 1998), leading to incomplete drying. If the droplets are not dry by the time they hit the wall of the spray tower, they will stick to the wall regardless of molecular weight of the maltodextrin due to the action of water as a plasticizer. Therefore, yields gained in a small laboratory scale spray tower are not a representation of what will occur in larger spray towers where the droplets have much longer time to dry before hitting the spray tower wall, and should be regarded with caution. 3.1.2

Water content and aw 10

ACCEPTED MANUSCRIPT Powders spray dried with maltodextrin DE 6 had a slightly higher water content (p0.05) than powders spray dried with maltodextrin DE 21 (within the same Tinlet /Toutlet ) (Table 1). However, there were no significant differences in aw between the powders (Table 1). Overall, the measured water content below 5% indicates a low-moisture product, and awvalues of 0.130.29 are in

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agreement with those established for food powders (aw = 0.200.60) (Roos, 2003). Potential

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stickiness of the powders is related to the amorphous or rubbery state of the wall material

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determined by its glass transition temperature (Tg). The formation of sticky powders can also

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occur 10-20C above the Tg, at which the wall material reaches a critical viscosity (Bhandari, & Howes, 1999; Roos, & Karel, 1991b). Spray drying with maltodextrin creates usually glassy

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amorphous structures (Descamps, Palzer, & Zuercher, 2009). Below Tg, the materials behave

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like solids (glass), and at and above Tg, the mobility of the molecules increase and the viscosity decreases, transitioning the material into a rubbery state (Roos, & Karel, 1991a). Maltodextrin

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DE 21 (on dry basis) has Tg between 134C (Ghorab, Toth, Simpson, Mauer, & Taylor, 2014)

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and 140C (Gianfrancesco, Turchiuli, Flick, & Dumoulin, 2010), whereas maltodextrin DE 6 (on dry basis) has Tg of 153C (Ghorab, et al., 2014) up to 180C. As Tg of both maltodextrins are

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well above the Toutlet of the spray drier (65 to 95C) (Table 1), the powders generated are

3.1.3

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therefore in an amorphous state, and not in the sticky region. Particle size of the redispersed powders

The liquid SLN and NLC samples before spray drying were nanosized (d 43 ~ 140 nm), which is in accordance with our earlier study (Salminen, et al., 2014a). The mean particle sizes of the redispersed spray dried lipid particles were affected by both maltodextrin type as well as by Tinlet /Toutlet (Table 1): The redispersed lipid particles spray dried with maltodextrin DE 6 were generally larger (d43 = ~300-800 nm) than those spray dried with maltrodextrin DE 21 (d43 = 11

ACCEPTED MANUSCRIPT ~180-435 nm) (Table 1). Redispersed SLN and NLC spray dried with maltodextrin DE 21 at Tinlet of 140C were the only particles with monomodal particle size distributions (Fig. A.1, Supplementary material). On the other hand, redispersed SLN and NLC at higher Tinlet showed bimodal particle distributions: The majority of the particles had a size around 180 nm, however,

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some bigger particles ranging between 1 to 40 m were also observed (Fig. A.1).

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In a previous study, spray dried powders of SLN coated with papain were reported to exhibit similar sizes as the ones before spray drying upon redispersion in isotonic model lung fluids

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(Gaspar, et al., 2017). Studies on nano-spray dried SLN stabilized by electrostatic layer-by-layer deposition of Na-caseinate and pectin showed that upon redispersion in water, the SLN showed

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rather good redispersibility, although the polydispersity index was higher (PDI = 0.27-0.35) than

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in the original liquid formulation (PDI = 016-0.22) (Wang, et al., 2016a; Xue, Wang, Hu, Zhou, & Luo, 2017). In another study, NLC loaded with fenofibrate, a drug for treating high cholesterol

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levels, was spray dried (Tinlet = 100C, Toutlet = 50-55C) with mixtures of mannitol and trehalose,

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and the redispersed powders had particle sizes of 0.74  0.11 m, whereas that of the NLC prior

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spray drying was around 0.1 m (Xia, Shrestha, van de Streek, Mu, & Yang, 2016). In general, the comparison of our results to literature is difficult, because multiple factors influence the

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outcome in spray drying including the feed composition (e.g., lipid type, wall material, solid concentration, and solution viscosity), spray drier type and dimensions (laboratory scale vs industrial scale), and spray drying conditions (e.g., feed rate, energy input, nozzle configuration, gas flow, humidity, Tinlet , Toutlet , drying rate, residence time, and aspirator rate) (BarbosaCánovas, Ortega-Rivas, Juliano, & Yan, 2005). 3.1.4

Thermal behavior

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ACCEPTED MANUSCRIPT The phase transition temperatures and crystal morphology of spray dried lipid particles was assessed by DSC. All spray dried lipid particles irrespective of the wall material and Tinlet showed two endothermic events at ~55-57C and ~69-71C that correspond to the melting of the - and -subcell crystals of tristearin, respectively (Figure 1a; data for NLC is shown as an

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example). Upon cooling from 85 to 10C, all the SLN and NLC samples exhibited a major

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crystallization peak at ~ 39 and 38C, respectively (Figure 1b; data for SLN not shown). In

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addition, in SLN and NLC samples spray dried with maltodextrin DE 6, small exothermic events

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starting around 47-44C were observed (Figure 1b; data for SLN not shown). Such exothermic events were, however, not detected with SLN samples spray dried with maltodextrin DE 21 (data

or

partially

coalesced,

thus

increasing

their

volume

(Helgason,

Kristinsson,

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coalesced

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not shown). This indicates that a small amount of bigger lipid particles generated by spray drying

Kristbergsson, & Weiss, 2011). This in turn increases the probability of finding more impurities

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in the particles, and therefore the lipid particles will crystallize at higher temperatures via

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heterogenous nucleation (Aquilano, & Squaldino, 2001), which typically occurs slightly below the TC of bulk tristearin (~52C).

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Next, we calculated the ratio between the melting enthalpy of -subcell crystals to the

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crystallization enthalpy (Hm()/HC) to compare the polymorphic state of the lipid particles (Table 1). An enthalpy ratio of one illustrates that the lipid particles remain fully in their subcell crystal form, whereas lower values indicate that the -subcell crystals are polymorphing into -subcell crystals. These results showed that SLN, regardless of the used maltodextrin type or applied Tinlet , retained almost fully their -subcell crystal structures (Table 1). In NLC, on the other hand, the enthalpy ratio after 1 day decreased dramatically. A general decrease in the

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ACCEPTED MANUSCRIPT enthalpy ratio was expected because liquid fish oil was incorporated into the structure, which leads to more random rearrangement of fatty acid chains (Awad, Helgason, Weiss, Decker, & McClements, 2009; Jenning, Thunemann, & Gohla, 2000; Salminen, et al., 2013). Nevertheless, the used Tinlet had a major impact on the polymorphic stability of the NLC by decreasing the

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Hm/HC -ratio with increasing Tinlet (Table 1). The impact of the maltodextrin type was less

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pronounced, however, maltodextrin DE6 as wall material showed slightly lower enthalpy ratios

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than NLC with maltodextrin DE 21 (Table 1).

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Based on the above results, we can conclude that the highest physical and polymorphic stability of NLC and SLN were achieved with maltodextrin DE 21 and by spray drying at Tinlet of

Storage stability of spray dried lipid particles

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3.2

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140C.

The objective of this series of experiments was to investigate the stability of spray dried solid

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lipid particles loaded with -3 fish oil during storage. All the storage experiments (Table 2)

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were carried out with maltodextrin DE 21 as it was shown to be a superior wall material to

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maltodextrin DE 6 in regard of the physical stability, i.e. the lipid particles spray dried with maltodextrin DE 6 showed larger and more polydisperse particle sizes upon redispersion

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indicating that these particles had a poorer physical stability (Table 1). Based on the results in chapter 3.1, we selected the best and worst spray drying conditions (Tinlet/outlet = 140/65C vs 170/95C) in terms of physical and polymorphic stability. We expected that the powders generated at Tinlet/outlet of 140/65C would perform better over time. As comparison, we also assessed the stability of the following samples: (i) spray dried SLN, (ii) unprocessed SLN and NLC dispersions (=liquid), and (iii) freeze dried SLN and NLC. In particular, we hypothesized that the spray dried solid lipid particles would exhibit improved physical and polymorphic 14

ACCEPTED MANUSCRIPT stabilities over freeze dried samples. This is because emulsions are often not stable against freezing (Freitas, & Müller, 1998; Ghosh, & Coupland, 2008). All the samples were stored at 35C in the dark over 71 days. Additionally, SLN and NLC powders spray dried at Tinlet of 140C were stored at 4C, which represented an optimal storage condition for the fish oil. Particle size of the redispersed powders

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3.2.1

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SLN and NLC dispersions (=liquid, not spray dried) remained nanosized (d43 150 nm) during the 71-day storage test (Figure 2). At storage temperature of 4C, SLN and NLC spray dried at

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Tinlet of 140C had particle sizes around 200 nm upon redispersion (Figure 2). Storage at higher

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temperature of 35C did not influence the particle size of NLC samples spray dried at Tinlet of 140C (p0.05) (Figure 2). On the other hand, SLN spray dried at Tinlet of 140C and stored at

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35C had larger particle sizes (d43 = 471  162 nm) compared to the ones stored at 4C (d43 = 197

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 31 nm) (Figure 2). The mean particle sizes for SLN and NLC at Tinlet of 170C were 584 151 nm and 389  80 nm, respectively, during the 71-day storage time (Figure 2). The larger particle

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sizes of NLC and SLN spray dried at Tinlet of 170C compared to samples at Tinlet of 140C

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(Figure 2) can be attributed to the melting of the tristearin carrier during spray drying. This means that the melted lipid droplets agglomerated to bigger lipid droplets before the

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maltodextrin formed a protective layer around the lipid particles. In contrast, the freeze dried SLN and NLC formed even larger and highly polydispersed particles (Figure 2). This is because the freezing step leads to formation ice crystals, which can then destabilize the surfactant layer on the particles (Freitas, & Müller, 1998) and lead to development of large agglomerated structures (Ali, & Lamprecht, 2017) as was also illustrated by a SEM-image (Figure 3). The SEM-image shows a massive block of material instead of spherical particles observed for the spray dried samples (Figure 3). Upon freeze drying, the 15

ACCEPTED MANUSCRIPT samples formed a hard cake that needed to be ground to a free-flowing powder. Moreover, the freeze dried powder became one bulk after 14 days of storage. This can be attributed to glass transition of the maltodextrin to its crystalline form at high water content (Bhandari, & Howes, 1999), which was determined to be ~10% for the freeze dried SLN and NLC.

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Based on these results, the storage temperature (4 or 35C) did not impact the particle sizes

Thermal behavior

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3.2.2

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of the NLC.

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Next, we plotted the Hm/HC -ratio as a function of storage time to gain insights into the polymorphic stability of the spray dried and freeze-dried samples (Figure 4). As explained above

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(chapter 3.1.4), the enthalpy ratios are generally lower in NLC because of the presence of liquid

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fish oil (Awad, et al., 2009; Jenning, Thunemann, & Gohla, 2000; Salminen, et al., 2013). The highest polymorphic stability was observed for SLN and NLC spray dried at Tinlet of 140C

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(stored at 4C), which retained 96 and 30% of their -subcell crystals, respectively (Figure 4).

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At storage temperature of 35C, SLN spray dried at Tinlet of 140 and 170C showed a similar retention of -subcell crystal morphology (60 versus 53%) after 71 days (Figure 4a). Similarly,

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the spray dried NLC (Tinlet = 140 and 170C) showed decreased Hm/HC -ratios upon storage

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at 35C: After 8 days of storage, only approximately 20% of the -subcell crystals were present, which further reduced to 10% after 71 days of storage (Figure 4b). The freeze dried SLN and NLC showed the poorest polymorphic stability and lost 96-98% of their -subcell crystals upon storage (Figure 4). This is in an agreement with the detection of large particle sizes (Figure 1) as already discussed in chapter 3.2.1. Interestingly, the Hm/HC -ratio also continuously decreased for SLN (0.37  0.00) and NLC (0.19  0.01) dispersions (i.e. no processing) during storage (Figure 4). This result is in 16

ACCEPTED MANUSCRIPT contrast to our previous results that showed that SLN and NLC stabilized by Quillaja saponins and high-melting lecithins are rather stable during storage, however, no maltodextrin was present in these samples (Salminen, et al., 2014a; Salminen, Gömmel, Leuenberger, & Weiss, 2016). Therefore, the addition of maltodextrin seems to have an impact on the polymorphic stability of

and

should

be investigated

more closely in further studies.

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polysaccharide molecules,

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the solid lipid particles, possibly due to interactions between the emulsifier layer and the

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Nevertheless, as the particle sizes of the “liquid” SLN and NLC increased only slightly during storage (Figure 2), we suggest that the amount of excess surfactant in the aqueous phase was

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enough to adsorb to the new surfaces created upon polymorphic transition (Helgason, et al.,

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2008; Helgason, Awad, Kristbergsson, McClements, & Weiss, 2009), and physically stabilize the particles.

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Overall, these results demonstrate that the storage temperature has a major impact on the

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polymorphic transition of spray dried solid lipid particles. Increasing the storage temperature has

(Helgason, et al., 2008).

Powder morphology and size

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3.2.3

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been shown to induce faster polymorphic transition from - to -subcell crystals in SLN

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The SEM-images revealed an uneven powder morphology with dents and concavities that is typical for spray dried powders (Figure 5). This can be attributed to the high temperatures that the particles experienced at the beginning of the drying process which led to particle shrinkage and rapid solidification of the wall material (Rosenberg, & Sheu, 1996; Shu, Yu, Zhao, & Liu, 2006). Similar behavior has been reported for liposomes spray dried with maltodextrin (Karadag, Özçelik, Sramek, Gibis, Kohlus et al., 2013) as well as for encapsulated monoterpenes (Bertolini, Siani, & Grosso, 2001) and flaxseed oil spray dried with gum arabic (Tonon, Grosso, &

17

ACCEPTED MANUSCRIPT Hubinger, 2011). Completely spherical particles without indentations are blown up by the drying gas as a crust is formed by the wall material (Serfert, et al., 2009). This effect is called “ballooning”. The curvatures and dents in the spray dried particles (Figure 5) indicate that no air is encased in the particles between the lipid and wall material. Only few particles, especially

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those spray dried at Tinlet of 170C, retained their fully spherical shape with smooth surfaces,

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however, these particles were hollow inside (Figure 5). This phenomenon with formation of air

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vacuoles which increases the surface lipids has been reported at higher outlet temperatures

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(Kelly, Kelly, & Harrington, 2002).

As illustrated by the SEM-images, the size of the powder particles was rather polydispersed

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varying from ca. 1 m above 10 m (Figure 5). Earlier studies have described that spray drying

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(Tinlet = 103C) of SLN with a papain coating for stabilization applying mannitol or trehalose as wall material generated powders with 5-7 m sizes (Gaspar, et al., 2017). Literature on spray

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drying of NLC coated with carrageenan or pectin reports also formation of ‘ultra-fine’ lipid

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powders, however, although the SEM-images indicated powder sizes ranging from 1 to 10 m (Wang, et al., 2016b). Studies using the layer-by-layer deposition of Na-caseinate and pectin on

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SLN have shown production of ultrafine ( 1 m) spray dried powders when applying a nano-

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spray drying technique (Tinlet = 100C) (Wang, et al., 2016a; Wang, Ma, Lei, & Luo, 2016; Xue, et al., 2017). Fenofibrate loaded NLC formed highly asymmetrical powders with 10 to100 m particle sizes upon spray drying (Tinlet = 100C, Toutlet = 50-55C) with mixtures of mannitol and trehalose (Xia, et al., 2016). In general, our results demonstrated that the morphology of the spray dried particles hardly changed upon application of Tinlet of 140 or 170C. Moreover, the storage time and elevated temperature also had no impact on the appearance or size of the powder particles. Any potential 18

ACCEPTED MANUSCRIPT changes in the internal powder structure or in the embedded solid lipid particles, on the other hand, could not be detected in these SEM micrographs. 3.3

Sensory analysis

The purpose of these experiments was to gain insights into the oxidative stability of the spray

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dried powders during 68 days of storage. According to the sensory evaluation, the spray dried

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SLN powders containing only saturated tristearin revealed a sweet and a slightly green and mouldy aroma that was neither impacted by the storage temperature (4 or 35C) (data not shown)

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nor storage time (Figure 6). These powders also remained whitish/grayish during the whole storage time (images not shown). The sensory panel assessed that the NLC spray dried at Tinlet of

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170C or freeze dried exhibited a somewhat fishier smell than those spray dried at Tinlet of 140C

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and was accompanied with green odors (Figure 6). Nevertheless, the statistical analysis revealed

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no significant differences. Upon storage at 4C, NLC spray dried at Tinlet of 140C showed no color changes in its white appearance during storage (images not shown). However, increasing

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the storage temperature to 35C was accompanied with a color change to a more

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yellowish/brownish (images not shown). An exposure to a higher temperature have been previously shown to increase the oxidation of encapsulated fish oil (Huang, et al., 2014).

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The fishy smell and the yellowish appearance of freeze dried NLC can be explained by the expulsion of fish oil out of the tristearin carrier upon freeze drying. The liquid fish oil, however, can still be partly entrapped in the amorphous maltodextrin structure right after freeze drying. Over time, the high water content enables the transition from the amorphous structure to a crystalline state in which molecular mobility increases and facilitates the release of volatile oxidation products, especially at elevated storage temperatures (To, & Flink, 1978). On the other hand, if the water content is low enough, the maltodextrin can remain in its glassy state and 19

ACCEPTED MANUSCRIPT functions as a barrier to lipid oxidation (Meste, Champion, Roudaut, Blond, & Simatos, 2002). It should be noted though that over time maltodextrin also tends to transition from the metastable glassy state into a more thermodynamically stable crystalline state (Bhandari, & Howes, 1999). Nonetheless, small molecules may still have some molecular mobility even in the glassy state

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that can result in exchange of volatile compounds (Meste, et al., 2002), especially if the aw

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exceeds a value of 0.3 (Velasco, Dobarganes, & Márquez-Ruiz, 2003). For example, diffusion of

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oxygen through an amorphous structure of lactose was reported to increase oxidation of methyl linoleate encapsulated into a mixture of lactose and gelatin by spray drying (Shimada, Roos, &

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Karel, 1991). At aw of 0.2-0.3, on the other hand, the mobility of molecules in the dried state is

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very limited which means that lipid oxidation also progresses very slowly (Velasco, Dobarganes, & Márquez-Ruiz, 2003). Our NLC samples showed aw-values of 0.23-0.33 (Table 1). Therefore,

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it is possible that the oxidation may have not proceeded far enough to show major differences. In

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conclusion, as no statistically significant differences between the NLC samples at 35C were

4.

KEY INSIGHTS

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observed, a more profound oxidation study should be carried out in the future.

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Our data showed that spray drying with maltodextrin DE 21 and at Tinlet/outlet of 140/65C

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produced the most physically and polymorphically stable lipid particles. We propose that when Toutlet is low enough (65C), the carrier lipid (tristearin) remained solid, and the SLN and NLC did not destabilize. Although this temperature is above the onset melting temperature of subcell crystals of tristearin (Tm(),onset = 53.4  0.9 C), it has been shown that particles may only experience temperatures of 15-20C below the Toutlet as most of the heat during spray drying is absorbed by the liquid surrounding the lipid particles (Freitas, & Müller, 1998). This means that the lipid particles may have eventually reached temperatures between 45 and 50C when dried at 20

ACCEPTED MANUSCRIPT Toutlet of 65C. In comparison, at Toutlet of 75-95C, the lipid particles experienced at least temperatures of 55 to 75C. Consequently, the solid lipid structures created prior spray drying retained their crystal structures also after spray drying when applying Tinlet /outlet of 140/65C, which indicated that the melting temperature of tristearin was not reached. This was verified by

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the particle size data (Table 1, Figure 2) that showed that the particle sizes remained small and

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monomodal upon redispersion, as well as by the DSC measurements (Figure 1, 4) that

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demonstrated that the lipid particles had also the highest polymorphic stability at Tinlet/outlet of

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140/65C. In contrast, at higher Tinlet (150-170C), the lipid particles melted during the spray drying process as was indicated by the generation of larger particles (Table 1, Figure 2) as well

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as by the poor polymorphic stability (Table 1, Figure 4).

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In the case of the maltodextrin type, one possible explanation for the better physical and

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polymorphic stability of the lipid particles when using maltodextrin DE 21 is that the lower molecular weight molecules can pack themselves more densely around the solid lipid particles,

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whereas maltodextrin DE 6 with high molecular weight molecules is more branched and more rigid, leaving the structure more randomly distributed. Such formation of voids in the polymer

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matrix depending on the type and molecular weight of the wall material upon spray drying have

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been reported (Drusch, Rätzke, Shaikh, Serfert, Steckel et al., 2009). This suggests that the highly packed maltodextrin DE 21 allows less movement in the solid lipid particles when it comes to the polymorphic transition. However, further studies are needed to confirm this. 5.

CONCLUSION

This study showed that the physical and polymorphic stability of food-grade SLN and NLC can be successfully retained upon spray drying as long as the chosen spray drying conditions do not exceed the melting temperature of the tristearin carrier. The best performance was achieved at 21

ACCEPTED MANUSCRIPT Tinlet/outlet of 140/65C and applying the low molecular weight maltodextrin (DE 21) as protective wall material. Further research in a pilot plant scale are underway. These results are promising basis for spray drying solid lipid particles for applications in food, feed, and pharmaceutical industries. ACKNOWLEDGEMENTS

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6.

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We thank DSM Nutritional Ltd. (Basel, Switzerland) for their financial support. We also thank

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Dipl. Food Sci. Eng. Martin Sramek (Department of Process Engineering and Food Powders,

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University of Hohenheim) for his support in spray drying, and Barbara Maier (Department of Food Physics and Meat Science, University of Hohenheim) for the scanning electron microscopy

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