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Microstructure and lactose crystallization properties in spray dried nanoemulsions
Accepted Manuscript Title: Microstructure and lactose crystallization properties in spray dried nanoemulsions Author: P.G. Maher M.A.E. Auty Y.H. Roos L.M. Zychowski M.A. Fenelon PII: DOI: Reference:
S2213-3291(14)00023-9 http://dx.doi.org/doi:10.1016/j.foostr.2014.10.001 FOOSTR 21
To appear in: Received date: Revised date: Accepted date:
17-7-2014 3-10-2014 9-10-2014
Please cite this article as: P.G. MaherM.A.E. AutyY.H. RoosL.M. ZychowskiM.A. Fenelon Microstructure and lactose crystallization properties in spray dried nanoemulsions (2014), http://dx.doi.org/10.1016/j.foostr.2014.10.001 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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*Graphical Abstract
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Maher Pg. 1 Highlights
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x Powders prepared from nanoemulsions crystallized more quickly than those prepared from conventional emulsions x Polarised light microscopy was used to confirm onset of crystallization and was consistent with dynamic vapour absorption data. x CLSM and cryo-SEM showed the distribution of fat globules within the particles in situ. Cryo-SEM was particularly useful for characterising nano-sized fat droplets (<200 nm) which were below the resolution limit of confocal microscopy. x It is suggested that reduced protein in the continuous phase with lactose contributed to the observed increase in crystallization rate in powders prepared from nanoemulsions
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Maher Pg. 2 12
Title: Microstructure and lactose crystallization properties in spray dried nanoemulsions
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P.G. Maher1, 2, M.A.E. Auty1*, Y.H. Roos2, L.M. Zychowski1, 2 M.A. Fenelon1,
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Fermoy, Co. Cork, Ireland and 2Food Technology, School of Food and Nutritional Sciences,
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University College Cork, Cork, Ireland.
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Food Chemistry & Technology Department, Teagasc Food Research Centre, Moorepark,
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Patrick G. Maher, Research Student, Food Chemistry & Technology Department, Teagasc
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Food Research Centre, Moorepark, Cork, Ireland.
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Mark A. Fenelon, Principal Research Officer, Food Chemistry & Technology Department,
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Teagasc Food Research Centre, Moorepark, Cork, Ireland.
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Yrjö H. Roos, Professor, School of Food and Nutritional Sciences, University College Cork,
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Cork, Ireland.
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Lisa M. Zychowski, Research Student, Food Chemistry & Technology Department, Teagasc
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Food Research Centre, Moorepark, Cork, Ireland.
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Mark A.E. Auty, Senior Research Officer, Food Chemistry & Technology Department,
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Teagasc Food Research Centre, Moorepark, Cork, Ireland.
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*Contact information for Corresponding Author:
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Dr. Mark Auty, Food Chemistry & Technology Department, Teagasc Food Research Centre,
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Moorepark, Fermoy, Co. Cork, Ireland. Tel: +3532542442.
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email: [email protected].
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Maher Pg. 3 Abstract
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The objective of this study was to characterize lactose crystallization behaviour and
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microstructure of spray dried nanoemulsions with different fat globule sizes (FGS). Powders
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of the same composition (57.7% w/w lactose, 12.3% w/w sodium caseinate, 27.7% w/w
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sunflower oil, and 2.3% w/w water) but different FGS (mean diameters 1100 nm and 160
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nm) prior to spray drying were manufactured. Differences in lactose crystallization were
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studied using dynamic vapour sorption (DVS) and polarized light microscopy (PLM).
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Crystallization kinetics was modelled using the Avrami and Yang equations. Results showed
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that lactose crystallized in three dimensions and more rapidly in powders with a smaller FGS.
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PLM images showed a higher rate of crystal formation for smaller FGS powders when stored
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for up to 4 days at 55% relative humidity. Confocal laser scanning microscopy (CLSM) and
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cryo-scanning electron microscopy (Cryo-SEM) images indicated the more evenly distributed
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small fat globules inside powder particles prepared from spray-dried nanoemulsions. The
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surface of powder particles was uneven and ruptured post lactose crystallization. Crystals of
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presumptive anhydrous D- and E-lactose (5:3 molar ratio) appeared after humidification.
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Results showed powder particles of the same composition were altered in lactose
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crystallization characteristics by changing the FGS of emulsions pre spray drying.
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Keywords: Lactose crystallization, dynamic vapour sorption, polarized light microscopy,
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confocal laser scanning microscopy, cryo-scanning electron microscopy.
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Highlights
63 x Reducing FGS of emulsions pre drying increased rate of lactose crystallization.
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x Crystallization occurred in three dimensions based on the Avrami exponent.
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x CLSM and cryo-SEM showed in situ smaller FGS in powders from nanoemulsions.
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x Needle-like anhydrous lactose crystals were observed by cryo-SEM and CLSM.
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x Particles were aggregated and lost their shape post lactose crystallization.
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1. Introduction
88 In dairy products, the degree of lactose crystallization has a significant effect on ingredient
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and food properties, such as texture and flavour. Crystallization of lactose in milk powders
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results in lumping and caking which has a negative impact on powder reconstitution (Lai and
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Schmidt, 1990). Component crystallization also causes the release of entrapped lipids in
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powders (Fäldt and Bergenståhl, 1996; Shimada et al., 1991) making them more susceptible
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to oxidation.
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Various methods can be used to quantify crystallization of amorphous sugars. Isothermal
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differential scanning calorimetry (DSC) was used by Roos and Karel (1990) and Kedward et
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al. (1998) to determine the crystallization of freeze dried lactose and sucrose at low water
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contents (< 5%) and high temperatures (365 – 400 K). X-ray diffraction (XRD) was used by
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Jouppila et al. (1997) and Miao and Roos (2005) to monitor crystallization kinetics of lactose,
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trehalose and lactose/trehalose mixtures. Mahlin et al. (2004) used atomic force microscopy
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(AFM) to quantify crystallization.
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Lactose crystallization can be measured by gravimetric means, by monitoring changes in
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mass of powder samples upon humidification (Iglesias and Chirife, 1978; Jouppila and Roos,
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1994; Lai and Schmidt, 1990). Crystallization results in sorbed water release, which is
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measured from mass decrease in a humidified powder (Burnett et al., 2004; Burnett et al.,
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2006). This method is most successfully used at ambient temperatures where the
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crystallization rate is quite slow and can be easily monitored (Kedward et al., 2000).
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Maher Pg. 6 Crystallization kinetics can be modelled using various relationships. The Avrami equation
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(Avrami, 1939, 1940) has been used by several researchers (Arvanitoyannis and Blanshard,
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1994; Haque and Roos, 2005; Kedward et al., 2000; Mazzobre et al., 2003). Derivative
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models from the Avrami equation include the Yang equation (Yang et al., 2010) and
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Urbanovici-Segal equation (Urbanovivi and Segal, 1990). The Urbanovici-Segal equation
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includes a parameter r (r > 0) that determines how far the model deviates from the Avrami
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equation (Soutari et al., 2012). Other kinetic models used to determine lactose crystallization
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are the Williams-Landel and Ferry (WLF) equation (Roos and Karel, 1991) and the Hoffman
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equation (Arvanitoyannis and Blanshard, 1994). The Arrhenius equation can be used to
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determine the activation energy (EA) required for crystallization (Schmidt et al., 1999). The
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Avrami equation determines the rate constant (k) and exponent (n) that, respectively,
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determine how fast the material crystallizes and in how many dimensions. Powders stored at
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increasing temperatures and humidities crystallize at faster rates (Soutari et al., 2012).
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Polarized light microscopy (PLM) is a useful technique in distinguishing crystalline from
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non-crystalline material (Hartel, 2001) and can be used to directly measure the rate of crystal
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growth. Mazzobre at al. (2003) used polarized light videomicroscopy (PLV), in conjunction
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with DSC, to determine the isothermal crystallization kinetics of lactose and lactose-trehalose
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mixtures. The PLV method proved a useful way of directly observing individual crystal
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growth and morphological aspects that were undetectable by DSC.
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The distribution of fat globules has a significant effect on functional properties of dairy
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powders (Pisecky, 1997). Techniques such as CLSM and cryo-SEM are useful in highlighting
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microstructural differences which help explain why dairy powders have various functional
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properties. CLSM is widely used for studying the microstructure of cheese, milk powder and
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chocolate (Auty et al., 2001). McKenna (1997) examined whole milk powder using CLSM.
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Maher Pg. 7 Kelly et al. (2014) used CLSM to analyse the distribution of fat droplets in powder samples
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where different fat blends were stabilized by sodium caseinate. Samples were dual-labelled
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with Nile Red / Fast Green FCF to show the distribution of fat and protein, respectively,
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throughout the powder particles. Air vacuoles were also visible in powders in images
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produced by CLSM. SEM techniques have been developed to examine the outer and inner
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structures of foods (Soottitantawat et al., 2003). SEM has been used in studies of
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microencapsulation properties of powder structures stabilized by sodium caseinate (Fäldt and
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Bergenståhl, 1996; Hogan et al., 2001a, b). Cryo-SEM is a useful technique in analyzing
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nano-sized particles embedded in food matrices (Dudkiewicz et al., 2011).
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The objectives of the present study were (1) to investigate the effects of reducing the FGS of
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emulsions prior to spray drying on water-induced lactose crystallization during storage; and
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(2) to characterize powder microstructure using correlative microscopy techniques (PLM,
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CLSM and cryo-SEM) to help explain differences of the physical properties of the powders.
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2. Materials and Methods
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2.1 Materials
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Two powders, containing 57.7% w/w lactose, 12.3% w/w sodium caseinate and 27.7% w/w
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sunflower oil, were used (Maher et al., (2014). The powders differed in their mean FGS as
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they were both homogenized (60 °C with a single pass) at different pressures prior to spray
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drying. Conventional emulsions were produced using in-line two-stage homogenization
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(model NS20006H, GEA Niro, Soavi, Parma, Italy) at first- and second-stage pressures of
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13.79 MPa and 3.45 MPa, respectively, giving a FGS of ~ 1100 nm. Nanoemulsions were
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produced by microfluidization with a Microfluidizer (model M-110EH, Microfluidics,
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Newton, MA, USA) at 100 MPa, giving a FGS of ~ 155 nm. All emulsions were spray dried
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Maher Pg. 8 at the same conditions (inlet/outlet dryer temperatures of 180 °C/80 °C) with a single stage
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pilot-scale spray dryer (Anhydro F1 Lab, Copenhagen, Denmark) equipped with a two-fluid
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nozzle atomization system (Type 1/8 JAC 316ss) and counter-flow drying. Resulting powders
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were labelled C and N for conventional emulsion powders (FGS ~ 1100 nm) and
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nanoemulsions powders (155 nm), respectively.
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2.2.1. Dynamic vapour sorption
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Water sorption isotherms of powders were obtained from the DVS Data Analysis Suite in
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Microsoft Excel using a DVS-1 analyser (Surface Measurement Systems, London, UK)
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equipped with a Cahn microbalance. Powder samples (~ 30 mg) were placed in the sample
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pan with an empty pan used as a reference. Samples were humidified at 55% relative
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humidity (RH) on the DVS for two days (48 h) for C and N at 25ºC. Mixing dry nitrogen gas
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(200 mL/min) with saturated water vapour in the correct proportion using mass flow
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controllers was used to get accurate RH readings. Samples were measured from three
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replicate spray drying trials.
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2.2.2. Polarized light microscopy
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Samples were examined using an Olympus BX51 light microscope (Olympus BX-51,
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Olympus Corporation, Tokyo, Japan) with a 10x dry objective lens using polarized light.
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Digital images (TIFF, 8-bit) were taken using a Jenoptik C14 Imagic camera, and captured.
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Powders were analysed pre- and post- lactose crystallization following storage over a
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saturated solution of Mg(NO3)2 (55% RH) for 4 days at 20 °C. Crystalline regions appeared
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as bright areas on the micrographs.
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Maher Pg. 9 2.2.3. Confocal laser scanning microscopy
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Visualization of powder particles was carried out using a Leica TCS SP5 confocal laser
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scanning microscope (CLSM; Leica Microsystems CMS GmbH, Wetzlar, Germany). Powder
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samples were placed onto a glass slide and labelled using a mixture of Fast Green and Nile
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Red or Nile Red alone (Auty et al., 2001). The dye mixture comprised Fast Green (aq. 0.01 g
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/ 0.1 L) and Nile Red dissolved in polyethylene glycol mol. wt. 400 (0.1g / 0.1 L) mixed in a
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ratio that both allowed diffusion of the dye molecules into the powder particles whilst
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maintaining particle integrity and preventing solubilisation. Based on trial and error, pre- and
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post-lactose crystallized powders were labelled, respectively, with 1:40 or 1:20 ratios of Fast
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Green to Nile Red. Dual excitation using 488nm/633nm was utilized for all Nile Red and Fast
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Green mixtures, while single excitation at 488 nm was used for the Nile Red images.
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Representative images of each sample were taken using 63x oil immersion objective with
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numerical aperture 1.4. Z-stacks were obtained in order to generate a three-dimensional
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structure of the particle and to identify surface fat staining. Red and, Green pseudo-coloured
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images (8-bit), 512 x 512 pixels in size, were acquired using a zoom factor of 1. A minimum
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of four z-stacks were taken per sample, with representative micrographs being shown.
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Samples were measured pre and post lactose crystallization (55% RH for 3 days).
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2.2.4. Cryo-scanning electron microscopy (Cryo-SEM)
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Cryo-SEM was used to examine the microstructure of powders. Powders were sprinkled onto
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the surface of OCT embedding compound (Agar Scientific Ltd, UK) on a slotted aluminium
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stub, then plunge-frozen into liquid nitrogen slush at
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to the cold stage using the Alto 2500 cryo-transfer device (Gatan Ltd., Oxford, UK). A cold
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scalpel was used to fracture samples. The sample was cooled to
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at
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Maher Pg. 10 imaging at !"#$ '=?Q
[
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Zeiss SMT Ltd., Cambridge, UK), operating at 2 kV and a working distance of 6 mm, was
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used to obtain secondary electron images. Representative micrographs were taken in order to
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visualize surface characteristics, including surface fat and lactose crystals, where present.
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Samples were analyzed pre- and post- lactose crystallization (55% RH for 3 days).
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Spray drying experiments were carried out in triplicate. One-way analysis of variance
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(ANOVA) was used with Minitab 15 (Minitab Ltd., Coventry, UK) to determine significant
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differences between lactose crystallization kinetics of both sets of powders by Fisher’s one-
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way multiple comparison test. Results were deemed statistically significant if P < 0.05.
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222 3. Results and discussion
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3.1 Water sorption kinetics
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Water sorption kinetics of powders C and N were measured by DVS at 55% RH and 25 °C.
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These conditions were chosen since Burnett et al. (2004) showed that below 30% RH lactose
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crystallization will not occur and above 58% RH lactose crystallization is nearly
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instantaneous.
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Figure 1a shows the mean water uptake in powders from 3 replicate samples on a solids-non-
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fat (SNF) basis over a 2880 min (48 h) period. It is shown that there was no difference in
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initial water sorption behaviour of the powders. The mean maximum sorption rate of water in
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the first 60 min was 7.4 g water / 100 g SNF for C, and 7.9 g water / 100 g SNF for N.
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Maximum water sorption occurred in approximately 140 min, upon which there was a
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decrease in mass and therefore evidence for onset of lactose crystallization (Burnett et al.,
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Maher Pg. 11 2004; Burnett et al., 2006). Powders C and N shown in Figure 1a crystallized at different
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rates, with lactose crystallising faster in N than in C. The mass of water decreased until full
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crystallization was achieved towards the end of the run. The mean crystallization rate of
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lactose in each powder showed 0.9 g water / 100 g SNF lost per hour for C and 1.9 g water /
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100 g SNF lost per hour for N (measured between 700 and 760 min). The onset (m0) and end
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points (m\) of lactose crystallization were found by calculating the intersection point of
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tangent lines to the respective baselines using the experimental data in Figure 1a. The mean
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onset points for C and N were 483 min and 424 min, respectively, showing that N started to
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crystallize before C. N also had a mean end point of 1098 min compared to a mean end point
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of 1240 min for C. This clearly showed that lactose crystallized faster in powders from
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nanoemulsions (674 min in total) than in powders from conventional emulsions (757 min in
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total). This result was similar to that of Maher at al. (2014) who found using Differential
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Scanning Calorimetry (DSC) that lactose in powders from nanoemulsions crystallized at
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significantly (P < 0.05) lower temperatures than in powders from conventional emulsions.
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Maher at al. (2014) explained that this was due to nanoemulsions having a smaller FGS
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which means that there was more surface area for protein to adsorb to, leaving less protein in
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the continuous lactose phase.
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More detailed quantitative analysis of lactose crystallization was achieved by converting the
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experimental data to the fraction crystallized over time, and subsequent results were analyzed
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statistically. This was calculated by comparing the mass of water absorbed by the powder at a
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given time (mt) to the mass of absorbed water at the end of crystallization (m\) and the mass
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of water absorbed at the onset of crystallization (m0). Then:
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Crystallization T t
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(1)
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Maher Pg. 12 The resulting isotherm is shown in Figure 1b and is the mean of 3 replicate powders of each
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type (C and N). The time has been normalized so that the onset of lactose crystallization is
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time zero and the y-axis has been normalized to reflect the crystallization fraction. Before
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lactose crystallization, the crystalline fraction was assumed to be zero and it was assumed to
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be one after crystallization. Various different equations have been used in literature to model
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crystallization kinetics, the most common of which is the Avrami model (1939):
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Tt
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where k is the rate constant (min-1) and n is the Avrami exponent. The rate constant (k)
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quantifies how fast the crystals grow indicating the stability of the system (Gaisford et al.,
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2009) and the Avrami exponent (n) defines the mechanism of crystallization. The Avrami
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rate constant mainly depends on the crystallization temperature (Kawamura, 1979) and
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typically follows an Arrhenius-type temperature dependency. It takes both nucleation and
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crystal growth into account (Sharples, 1966). An Avrami exponent value of 1 represents
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nucleation and crystal growth from surfaces, a value of 2 represents crystal growth from
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edges, and a value of 3 indicates crystal growth in three dimensions. Non-integer n values can
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also be obtained and they represent mixed crystallization mechanisms (Cahn, 1956; Soutari et
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al., 2012). The sigmoidal shape of the curve in Figure 1b is typical of those produced using
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the Avrami equation. The larger the Avrami exponent (n) the more sigmoidal the
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crystallization curve (Avrami, 1940). Transformation rates are low at the beginning and end
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of the transformation, but rapid in between. Initially there is a slow rate due to the time
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required to form a significant number of nuclei of the new phase. The nuclei grow rapidly
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into particles, during the intermediate phase, and consume the old phase while nuclei
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continue to form in the remaining parent phase. When the transformation is nearly complete,
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there is less material to transform to nuclei and the production of new particles becomes slow.
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Also, the already existing particles touch each other forming a boundary where growth stops.
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(2)
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Maher Pg. 13 This is not accounted for in the Avrami model as it assumes a constant crystal growth rate,
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which is why the model breaks down as complete crystallization approaches (Soutari et al.,
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2012).
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The experimental data was also modelled using a modified version of the Avrami equation
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from Yang et al. (2010):
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Tt
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The same equation was derived by Tobin et al. (1974). Crystallization isotherms were fitted
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to Eqs. (2) and (3) and the results were compared to the experimental data in Table 1. The
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Avrami model was found to better describe the experimental crystallization process than the
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Yang model. The mean Avrami rate constants of 1.1 x 10-8 min-1 (C) and 1.2 x 10-7 min-1 (N)
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are significantly different (P < 0.05) showing that lactose in powders from nanoemulsions
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crystallized faster than in conventional emulsions (Table 1). The Avrami integers are very
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close to 3 indicating the crystals are growing in three dimensions (Soutari et al., 2012).
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Half-times of lactose crystallization are useful in predicting the storage stability of lactose
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containing products (Jouppila et al., 1997). The crystallization half-time is calculated using
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the rate constant (k) and exponent (n) in the following equation:
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(3)
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1/ n
§ ln 2 · ¨ ¸ © k ¹
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t1/ 2
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The t1/2 values, calculated using the Avrami k and n values, were very similar to those found
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experimentally (Table 1). High coefficients of determination (r2) and similar t1/2 values
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indicate that the Avrami equation models the experimental data to a high degree of accuracy.
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Figure 1b shows that Avrami crystallization values are very similar to experimental values.
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Values found using the Yang equation were less accurate than those from the Avrami
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equation (Table 1). The coefficient of determination was lower and the t1/2 was much
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different to the experimental value. The exponents (n) of 4.39 (C) and 4.31 (N) were greater
(4)
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Maher Pg. 14 and rate constants (k) were lower than Avrami constants. Similar results were reported by Al-
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Mulla (2007) who found that the Yang (or Tobin) model did not fit as well as the Avrami
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model because it gave higher n values and lower r2 values for the crystallization of polymers.
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The rate constants (k) obtained in this study (1.1 – 12 x 10-8 min-1) were much lower than
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those obtained by Schmidt et al. (1999), who measured the crystallization kinetics of
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amorphous lactose at 25 °C in the range 52.5 – 57.5% RH (5.6 – 15.5 x 10-3 min-1). Jouppila
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et al. (1997) found that the Avrami model did not work for skimmed milk powder (SMP) at
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53.8% RH, but did work at humidities ]^^!_!` ${? [!
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x 10-10 min-1) with a corresponding increase in crystallization half-time (40 h) compared to
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the powders in this study, even at the higher RH of 66.2%. Similar Avrami indexes were
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obtained for SMP (~ 3), indicating crystal growth in three dimensions. Overall, this shows
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that protein had a significant effect on lactose crystallization kinetics. When protein was not
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present, lactose crystallized quickly (Schmidt et al., 1999). The 11% sodium caseinate in this
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study caused delayed lactose crystallization, and increasing the protein content to 35% (for
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SMP) further delayed lactose crystallization (Jouppila et al., 1997). It is postulated that
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nanoemulsions have less non-adsorbed protein than conventional emulsions due to their
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smaller FGS giving increased surface area for protein adsorption. Therefore, this study
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suggests that not only is the total amount of protein important, but the amount of non-
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adsorbed protein that has the most significant effect on lactose crystallization.
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To the author’s best knowledge no other studies have compared crystallization of lactose in
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powders with significantly different FGS. The best comparison can be made with McCarthy
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et al. (2013) who showed that lactose crystallization occurred at lower ware contents with
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decreasing protein content (i.e., less protein in the continuous phase) for infant formula
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powders. Similar results were found by Thomas et al. (2004) who found that increasing E-
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lactoglobulin contents increased the humidity at which lactose crystallized in E-
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lactoglobulin/lactose mixtures.
335 3.2 Microscopy
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3.2.1 Polarized light microscopy
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Figure 2 shows PLM images of powders C and N when stored in a desiccator over a saturated
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salt solution of Mg(NO3)2 at 55% RH for 0, 1, 2, 3, and 4 days. The micrographs are
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consistent with results obtained from DVS, showing that lactose crystals formed more
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quickly in powder N than powder C over 4 days. Both powders at day 0 showed no evidence
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of lactose crystals. This was in contrast to other studies (Haque and Roos, 2005; McCarthy et
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al., 2013; Roetman, 1979) that showed lactose crystals present in fresh powders. At day 1, no
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crystals were seen in powder C but some small isolated crystals were seen in powder N
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(Figure 2d). At day 2, some crystals had formed in powder C (Figure 2e) with increased
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growth of crystals on the edges of powder particles in powder N (Figure 2f). Surface crystals
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are evident in powder C at day 3 (Figure 2g) but there are noticeably more crystals present in
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powder N (Figure 2h). By day 4, both powders C and N showed crystallization throughout
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the particles.
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3.2.2. Confocal laser scanning microscopy
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Confocal laser scanning microscopy results are shown in Figure 3. Labelling with Nile
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Red/Fast Green highlighted the fat and protein phases of the powder and showed air vacuoles
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as dark regions within the particle (Figure 3a, b, arrows). Discrete spherical fat droplets, most
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of which are 1 - 5 Pm in diameter, were clearly seen within powder C (Figure 3a). By
356
contrast, fat droplets in Powder N were small (Figure 3b); although some individual droplets
357
(~ 0.2 - 1Pm diameter) were visible, there was considerable co-localization of the Nile Red
Page 16 of 33
Maher Pg. 16 and Fast Green signals indicating that most fat droplets were below the resolution limit of the
359
CLSM. The protein phase of both powders is shown in red. The protein is more clearly
360
observed in powder C than powder N due to the fat globules being less numerous and evenly
361
spaced, allowing more gaps for the protein phase to be visualized. Very little surface fat was
362
visible for either powder (Figure 3a, b), in contrast with some agglomerated whole milk
363
powder particles that have shown evidence of surface fat pooling (McKenna, 1997). No
364
evidence of lactose crystals was found in fresh powders (Figure 3a, b).
cr
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358
us
365
Powders stored after 4 days at 55% RH are shown in Figure 3 c, d, e, f. Particles from both
367
powders appeared to have deformed and were more agglomerated than fresh powders. Figure
368
3c shows large free fat droplets (arrows) > 5 Pm in diameter as well as fat droplets and pools
369
within the particles. Powder N shows generally less free fat but had more obvious elongated
370
or needle-shaped crystals seen by negative contrast (Figure 3d, arrows). The protein signal
371
was fairly weak and appeared localized at the surface and outer edges of the particles (Figure
372
3 c, d). This may be due to high level of lactose crystallization preventing the stain from
373
diffusing into the particle core.
M
d
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374
an
366
375
To study the fat phase in more detail and reduce possible staining-induced coalescence,
376
powders were stained with Nile Red solubilized in polyethylene glycol, mol. wt. 400 and
377
results are shown in Figure 3e, f. The fat globules within the particles no longer appear
378
spherical as in fresh powders, probably due to lactose crystallizing around them and forcing
379
them into irregular pools (see inset Figure 3e). Powder N appeared to have less free fat and
380
more intact air vacuoles, indicating that powder particles from nanoemulsions are more stable
381
to crystallization-induced fat rupture.
382
Page 17 of 33
Maher Pg. 17 Few studies have looked at powders using CLSM post lactose crystallization. McCarthy et al.
384
(2013) used CLSM and found free fat partially covering lactose crystals post crystallization
385
of a model infant formula after it had leaked from the interior of the powder. McKenna
386
(1997) showed that for whole milk powder, when stained for fat only, that the entire powder
387
particle could be seen with lactose crystals clearly evident from negative contrast. The lactose
388
crystals in that study were rhomboidal D-crystals due to the lactose crystallizing in the milk
389
prior to spray drying. Needle-shaped crystals were seen post lactose crystallization in the
390
current study. Jouppila et al. (1997) and Bushill et al. (1965) reported that lactose in skimmed
391
milk powder crystallized at 55% RH in a 5:3 molar ratio of anhydrous D-lactose: E-lactose,
392
therefore our results are assumed to be the same.
an
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383
M
393 3.2.3 Cryo-scanning electron microscopy
395
It is important to characterize the outer topography of particles as it can give information on
396
flow properties of spray dried powders (Soottitantawat et al., 2003). Cryo-SEM images are
397
presented in Figures 4 and 5. The surface morphology of fresh powders from C and N
398
powders is shown in Figures 4a and b, respectively. The surfaces of all powders appeared to
399
be very smooth with little indentation and powder rupture, similar to data of Hogan et al
400
(2001b) who also used sodium caseinate as an emulsifier. Sodium caseinate is a very efficient
401
emulsifier and dominates the powder surface, even in small concentrations, due to a higher
402
surface activity of protein in water during the drying process (Fäldt and Bergenståhl, 1996).
403
Very little solvent extractable free fat was measured previously by Maher et al. (2014), with
404
powder N (0.4%) having a significantly lower FF content than powder C (2.3%). There was
405
little evidence of free fat on the surface of these powders. Fractured particles are shown in
406
Figure 4c, d, e, f. The advantage of cryo-SEM is that the air vacuoles are easily visible and
407
well preserved. Both powders showed high levels of occluded air in the form of rounded
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Page 18 of 33
Maher Pg. 18 408
vacuoles (Figure 4c, d, arrows), although powder N appeared to have slightly larger vacuoles
409
and thinner cross-walls than powder N. Vacuoles were distributed evenly throughout the
410
powder particles and were mostly in the size range 5 – 50 Pm.
ip t
411 Conventional emulsion powders (C) have an uneven distribution of oil droplets spread
413
throughout the powder particles, whereas nanoemulsion powders (N) have an even
414
distribution of much smaller oil droplets. The size of the oil droplets was ~ 1100 nm for C
415
and ~ 150 nm for N. The smaller oil droplets in nanoemulsions were more embedded in the
416
matrix and therefore less likely to be extracted upon addition of solvent. No evidence of
417
lactose crystals was found in any of the fresh powders. Higher magnifications revealed the
418
powder wall structure at the nano-scale (Figure 4e, f, g, h). In powder C spherical fat
419
droplets, mostly 200 nm to 5 Pm in diameter, are seen embedded within the wall matrix
420
(Figure 4 e, f, arrows). Fat droplets were smaller in powder N (< 200 nm diameter) and could
421
only be seen at very high magnifications (Figure 4h, arrow).
us
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422
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412
The powders were also examined by cryo-SEM post lactose crystallization (Figure 5). At low
424
magnification, agglomeration (or melding) of powder particles was evident (Figure 5a, b).
425
Higher magnification revealed the presence of radiating clusters of needle-like crystals on
426
powder surfaces (Figure 5c, d). Surfaces of lactose-crystallized powders were more uneven
427
with various indentations and holes. The irregular surface, as a result of lactose
428
crystallization, was also observed by Murrieta-Pazos et al. (2011). Faldt and Bergenståhl
429
(1996) found using electron spectroscopy for chemical analysis (ESCA) that a protein film
430
dominates the powder surface even after lactose crystallization. Solid bridges formed
431
between particles after lactose crystallization making them agglomerate and cluster, which is
432
in contrast to the more discrete individual particles from the fresh powder. No large crystals
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423
Page 19 of 33
Maher Pg. 19 were observed on the powder surface. Faldt and Bergenståhl (1996) postulated that fat
434
globules reduce the size of lactose crystals formed after humidification. Fractured particles
435
showed crystals embedded within the wall and also projecting into vacuoles (Figure 5e, f,
436
arrows). There was little evidence of intact spherical fat droplets in either powder, suggesting
437
that they had been disrupted by lactose crystals. This observation is consistent with CLSM
438
results which showed irregular fat pools dispersed within the powder particles (Figure 3e)
439
and highlights the advantage of a correlative microscopy approach. Cryo-SEM had the
440
advantages of good structural preservation and high resolution but could not easily
441
distinguish between fat, protein or amorphous lactose. CLSM revealed the distribution of fat
442
in powders more clearly, while PLM revealed the onset and progress of crystallization over
443
time and confirmed DVS data which indicated that powders prepared from nanoemulsions
444
crystallized at a faster rate. Combining cryo-SEM with CLSM and PLM thus provided a
445
powerful set of tools for characterising the microstructures of dairy powders.
446
te
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an
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cr
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433
Conclusions
448
Powders prepared from nanoemulsions crystallized significantly (P < 0.05) more quickly
449
than those prepared from conventional emulsions, as measured using DVS. Rates were
450
calculated with the Avrami equation and crystals were found to form in three dimensions.
451
Evidence of crystal growth was observed with PLM and showed the same trend as results
452
from DVS. CLSM and cryo-SEM show differences in powder structure with powders from
453
nanoemulsions having smaller fat globules that are evenly distributed throughout powder
454
particles. It is suggested that reduced protein in the continuous phase with lactose contributed
455
to observed increase in crystallization rate in powders prepared from nanoemulsions.
456
Elongated lactose crystals (probably a mixture 5:3 molar ratio of D and E-lactose), initially
457
at the surface but progressively occurring within the particles, were observed post
Ac ce p
447
Page 20 of 33
Maher Pg. 20 humidification at 55% RH. To the authors’ knowledge, there have been no studies on
459
powders prepared from nanoemulsions using CLSM, SEM, or cryo-SEM that show the
460
distribution of fat globules within the particles in situ. This paper provides a useful insight
461
into how reducing the FGS of emulsions pre spray drying impacts upon the physical
462
properties of powders.
ip t
458
cr
463 Acknowledgements
465
The authors would like to acknowledge the National Food Imaging Centre, Teagasc Food
466
Research Centre, Moorepark, Fermoy, Co. Cork, Ireland for use of the microscopes. The
467
project was funded by the Irish Department of Agriculture, Food and the Marine under the
468
Food Institutional Research Measure (FIRM).
M
469
479
d
Industrial relevance
te
Relatively little is known about the physical and chemical properties of nano-sized emulsions when spray dried. Drying of milk-based food ingredients is a major industry and high-shear processing is increasingly used to reduce fat droplet size to improve stability. This paper highlights one unintended consequence of reducing fat droplet size, namely increasing the tendency for lactose to crystallize. This should be factored in to any future formulations that involve high shear processing of milk derived ingredient s for subsequent spray drying.
Ac ce p
470 471 472 473 474 475 476 477 478
an
us
464
Page 21 of 33
Maher Pg. 21 479 480
Table 1 Summary of kinetic parameters obtained by fitting the crystallization isotherms to the
481
Avrami model and Yang model.
ip t
482 483
n
t1/2
r2
k
(min-1)
-
(min)
-
(min-1)
C
1.1 x 10-8 a
3.06 a
363 a
0.995
7.3 x 10-12 a
N
1.2 x 10-7 b
2.76 b
287 b
0.985
2.3 x 10-9 a
485
a-b
486
way ANOVA.
490 491 492 493 494
r2
t1/2
-
(min)
-
(min)
4.39 a
323 a
0.976
368 a
4.31 a
262 b
0.970
285 b
d
M
Values within a column not sharing a common letter differ significantly (P < 0.05) by one-
te
489
t1/2
Ac ce p
488
Experimental
n
an
k
Powder
487
Yang
us
Avrami
cr
484
495 496 497 498 499
Page 22 of 33
Maher Pg. 22 500 Figure captions
502
Figure 1. a) Dynamic vapour sorption graph of control (C) and nanoemulsion (N) powders
503
held at 55% RH for 24 h showing lactose crystallization. b) Crystallization isotherms for C
504
and N at 25 °C for experimental data and those calculated using the Avrami equation.
506
Figure 2. Polarized light (crossed polars) micrographs of control (a,c,e,g,i) and nanoemulsion
507
(b,d,f,h,j) powders. after storage for 0 (a,b), 1 (c,d), 2 (e,f), 3 (g,h) and 4 (i,j) days at 55%
508
relative humidity. Arrows indicate small crystals, scale bar = 100 Pm.
an
us
cr
505
ip t
501
509
Figure 3. Confocal scanning laser micrographs of showing internal microstructures of control
511
(a,c,e) and nanoemulsion (b,d,f) powders after storage for 0 (a,b) and 4 (c,d,e,f) days at 55%
512
relative humidity. Figure 4 a – d are fluorescently labelled with Nile red/fast green to show
513
fat (green) and protein (red); Figure 4 e & f are labelled with Nile Red to show fat phase.
514
Arrows indicate air vacuoles (a, b), discrete fat droplets (c) and solid lactose crystals (d). Fig
515
3e inset shows irregularly shaped coalesced fat regions. Scale bars = 100 Pm.
d
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Ac ce p
516
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510
517
Figure 4. Cryo-scanning electron micrographs of control (a,c,e,g) and nanoemulsion (b,d,f,h)
518
powders prior to crystallization. Images show powder particle surface morphology (a,b) and
519
interior features of fractured particles (c – h). Arrows indicate air vacuoles (c,d) and fat
520
droplets (e,g,h). Scale bars = 20 Pm (a,b), 10 Pm (c,d), 2 Pm (e,f) and 500 nm (g,h).
521 522
Figure 5. Cryo-scanning electron micrographs of control (a,c,e) and nanoemulsion (b,d,f)
523
powders after storage for 4 days at 55% relative humidity showing crystals (arrowed) on the
Page 23 of 33
Maher Pg. 23 powder surfaces (a,b,c,d) and also interior of fractured particles. Scale bars = 20 Pm (a,b), 5
525
Air vacuole Pm (c,d) and 2 Pm (e,f).
526
Figures
Ac ce p
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an
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524
527 528
Figure 1.
529 530
Page 24 of 33
Maher Pg. 24 531 532 533
a
b
c
d
e
f
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534 535
cr
536
538
us
537
an
539 540
te
544 545 546 547 548
g
549 550 551 552
i
Ac ce p
543
d
542
M
541
h
j
553 554 555
Page 25 of 33
Maher Pg. 25 556
558 559
Figure 2.
a
b
c
d
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557
560
cr
561 562
us
563
an
564
566
M
565
567
d
568
te
569
571 572 573 574 575 576
Ac ce p
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e
f
577 578 579 580
Page 26 of 33
Maher Pg. 26 581 582 583
Figure 3.
a
b
c
d
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584 585
cr
586
589
an
588
590
M
591 592
597 598 599 600 601
Ac ce p
595 596
f
te
e
d
593 594
us
587
g
h
602 603 604
Figure 4.
605
Page 27 of 33
Maher Pg. 27 606 607 608
a
b
c
d
ip t
609 610
cr
611 612
an
614 615
M
616 617
d
618
e
f
te
619
622 623 624 625 626
Ac ce p
620 621
us
613
Figure 5.
627 628 629 630
Page 28 of 33
Maher Pg. 28 631 632 References
634
Al-Mulla, A., (2007). Isothermal crystallization kinetics of poly(ethylene terephthalate) and
635
poly(methyl methacrylate) blends. eXPRESS Polymer Letters 1(6), 334-344.
636
Arvanitoyannis, I., Blanshard, J.M.V., (1994). Rates of Crystallization of Dried Lactose-
637
sucrose Mixtures. Journal Of Food Science 59(1), 197-205.
638
Auty, M.A.E., Twomey, M., Guinee, T.P., Mulvihill, D.M., (2001). Development and
639
application of confocal scanning laser microscopy methods for studying the distribution of fat
640
and protein in selected dairy products. Journal of Dairy Research 68, 417-427.
641
Avrami, M., (1939). Kinetics of phase change. I. General theory. Journal of Chemical Physics
642
7, 1103-1112.
643
Avrami, M., (1940). Kinetics of Phase Change II. Transformation-Time Relations for
644
Random Distribution of Nuclei. Ibid 8, 212-224.
645
Burnett, D., Thielmann, F., Booth, J., (2004). Determining the critical relative humidity for
646
moisture-induced phase transitions. International Journal of Pharmaceutics 287, 123-133.
647
Burnett, D., Thielmann, F., Sokoloski, T., Brum, J., (2006). Investigating the moisture-
648
induced crystallization kinetics of spray-dried lactose. International Journal of Pharmaceutics
649
313, 23-28.
650
Bushill, J.H., Wright, W.B., Fuller, C.H.F., Bell, A.V., (1965). The crystallisation of lactose
651
with particular reference to its occurrence in milk powder. Journal of the Science of Food and
652
Agriculture 16(10), 622-628.
653
Cahn, J.W., (1956). Transformation kinetics during continuous cooling. Acta Metallurgia 4,
654
572-575.
Ac ce p
te
d
M
an
us
cr
ip t
633
Page 29 of 33
Maher Pg. 29 Dudkiewicz, A., Tiede, K., Loeschner, K., Jensen, L.H.S., Wierzbicki, R., Boxall, A.B.A.,
656
Molhave, K., (2011). Characterization of nanomaterials in food by electron microscopy.
657
Trends in Analytical Chemistry 30(1), 28-43.
658
Fäldt, P., Bergenståhl, B., (1996). Changes in Surface Composition of Spray-Dried Food
659
Powders due to Lactose Crystallization. LWT - Food Sci. Technol. 29(5–6), 438-446.
660
Gaisford, S., Verma, A., Saunders, M., Royall, P.G., (2009). Monitoring crystallisation of
661
drugs from fast-dissolving oral films with isothermal calorimetry. International Journal of
662
Pharmaceutics 380, 105-111.
663
Haque, M.K., Roos, Y.H., (2005). Crystallization and X-ray diffraction of spray-dried and
664
freeze-dried amorphous lactose. Carbohydrate Research 340, 293-301.
665
Hartel, R., (2001). Measurements of crystalline structure in foods. In: Crystallization in
666
Foods. Aspen Publishers: Gaithersburg, MD, p 34-90.
667
Hogan, S.A., McNamee, B.F., O'Riordan, E.D., O'Sullivan, M., (2001a). Emulsification and
668
microencapsulation properties of sodium caseinate/carbohydrate blends. International Dairy
669
Journal 11(3), 137-144.
670
Hogan, S.A., McNamee, B.F., O'Riordan, E.D., O'Sullivan, M., (2001b). Microencapsulating
671
Properties of Sodium Caseinate. Journal of Agricultural and Food Chemistry 49, 1934-1938.
672
Iglesias, H.A., Chirife, I., (1978). Delayed crystallization of amorphous sucrose in humidified
673
freeze dried model systems. Journal of Food Technology 13, 137-144.
674
Jouppila, K., Kansikas, J., Roos, Y.H., (1997). Glass transition, water plasticization, and
675
lactose crystallization in skim milk powder. J Dairy Sci 80(12), 3152-3160.
676
Jouppila, K., Roos, Y.H., (1994). Glass transitions and crystallization in milk powders. J
677
Dairy Sci 77(10), 2907-2915.
678
Kawamura, K., (1979). The DSC Thermal Analysis of Crystallization Behaviour in Palm Oil.
679
Ibid 56, 753-758.
Ac ce p
te
d
M
an
us
cr
ip t
655
Page 30 of 33
Maher Pg. 30 Kedward, C.J., MacNaughtan, W., Blanshard, J.M.V., Mitchell, J.R., (1998). Crystallization
681
Kinetics of Lactose and Sucrose On Isothermal Differential Scanning Calorimetry. Journal Of
682
Food Science 63(2), 192-197.
683
Kedward, C.J., MacNaughtan, W., Mitchell, J.R., (2000). Isothermal and non-isothermal
684
crystallization in amorphous sucrose and lactose at low moisture contents. Carbohydrate
685
Research 329(2), 423-430.
686
Kelly, G.M., O’Mahony, J.A., Kelly, A.L., O’Callaghan, D.J., (2014). Physical
687
characteristics of spray-dried dairy powders containing different vegetable oils. Journal of
688
Food Engineering 122(0), 122-129.
689
Lai, H., Schmidt, S., (1990). Lactose crystallisation in skim milk powder observed by
690
hydrodynamic equilibria, scanning electron micrroscopy and 2H nuclear magnetic resonance.
691
J Food Sci 55(4), 994-999.
692
Maher, P.G., Roos, Y.H., Fenelon, M.A., (2014). Physicochemical properties of spray dried
693
nanoemulsions with varying final water and sugar contents. J. Food Eng. 126(0), 113-119.
694
Mahlin, D., Berggren, J., Alderborn, G., Engstrom, S., (2004). Moisture-Induced Surface
695
Crystallization of Spray-Dried Amorphous Lactose Particles Studied by Atomic Force
696
Microscopy. Journal of Pharmaceutical Sciences 93(1), 29-37.
697
Mazzobre, M.a.F., Aguilera, J.M., Buera, M.a.P., (2003). Microscopy and calorimetry as
698
complementary techniques to analyze sugar crystallization from amorphous systems.
699
Carbohydrate Research 338(6), 541-548.
700
McCarthy, N.A., Gee, V.L., Hickey, D.K., Kelly, A.L., O'Mahony, J.A., Fenelon, M.A.,
701
(2013). Effect of protein content on the physical stability and microstructure of a model
702
infant formula. International Dairy Journal 29(1), 53-59.
703
McKenna, A.B., (1997). Examination of whole milk powder by confocal laser scanning
704
microscopy. Journal of Dairy Research 64, 423-432.
Ac ce p
te
d
M
an
us
cr
ip t
680
Page 31 of 33
Maher Pg. 31 Miao, S., Roos, Y.H., (2005). Crystallization Kinetics and X-ray Diffraction of Crystals
706
Formed in Amorphous Lactose, Trehalose, and Lactose/Trehalose Mixtures. Journal Of Food
707
Science 70(5), E350-E358.
708
Murrieta-Pazos, I., Gaiani, C., Galet, L., Cuq, B., Desobry, S., Scher, J., (2011). Comparative
709
study of particle structure evolution during water sorption: Skim and whole milk powders.
710
Colloids and Surfaces B: Biointerfaces 87(1), 1-10.
711
Pisecky, J., (1997). Handbook of milk powder manufacture, Copenhagen.
712
Roetman, K., (1979). Crystalline lactose and the structure of spray-dried milk products as
713
observed by scanning electron microscopy. Netherlands Milk and Dairy Journal 33, 1-11.
714
Roos, Y., Karel, M., (1990). Differential scanning calorimetry study of phase transitions
715
affecting the quality of dehydrated materials. Biotechnology Progress 6(2), 159-163.
716
Roos, Y.H., Karel, M., (1991). Plasticizing Effect of Water on Thermal Behaviour and
717
Crystallization of Amorphous Food Models. J Food Sci 56(1), 775-777.
718
Schmidt, E.A., Law, D., Zhang, G.G.Z., (1999). Nucleation and Crystallization Kinetics of
719
Hydrated Amorphous Lactose above the Glass Transition Temperature. Journal of
720
Pharmaceutical Sciences 88(3), 291-296.
721
Sharples, A., (1966). Overall Kinetics of Crystallization. In: Sharples, A., editor. Introduction
722
to Polymer Crystallization, Edward Arnold Ltd, London, p 44–59.
723
Shimada, Y., Roos, Y., Karel, M., (1991). Oxidation of methyl linoleate encapsulated in
724
amorphous lactose-based food model. Journal of Agricultural and Food Chemistry 39(4),
725
637-641.
726
Soottitantawat,
727
Microencapsulation by spray-drying: Influence of emulsion size on the retention of volatile
728
compounds. J. Food Sci. 68(7), 2256-2262.
Ac ce p
te
d
M
an
us
cr
ip t
705
A.,
Yoshi,
H.,
Furata,
T.,
Ohkawara,
M.,
Linko, P.,
(2003).
Page 32 of 33
Maher Pg. 32 Soutari, N., Buanz, A.B.M., Gul, M.O., Tuleu, C., Gaisford, S., (2012). Quantifying
730
crystallisation rates of amorphous pharmaceuticals with dynamic mechanical analysis
731
(DMA). International Journal of Pharmaceutics 423, 335-340.
732
` {!!!$ ! $!['! -Lactoglobulin Interaction During
733
Storage of Model Whey Powders. Journal of Dairy Science 87(5), 1158-1166.
734
Tobin, M.C., (1974). Theory of phase transition kinetics with growth site impingement. I.
735
Homogenous nucleation. Journal of Polymer Science Part B: Polymer Physics 12, 399-406.
736
Urbanovivi, E., Segal, E., (1990). New formal relationships to describe kinetics of
737
crystasllisation. Thermochimica Acta 171, 87-94.
738
Yang, J., Grey, K., Doney, J., (2010). An improved kinetics approach to describe the physical
739
stability of amorphous solid dispersions. International Journal of Pharmaceutics 384(1–2),
740
24-31.
an
M te
744
Ac ce p
743
d
741 742
us
cr
ip t
729
Page 33 of 33
S2213-3291(14)00023-9 http://dx.doi.org/doi:10.1016/j.foostr.2014.10.001 FOOSTR 21
To appear in: Received date: Revised date: Accepted date:
17-7-2014 3-10-2014 9-10-2014
Please cite this article as: P.G. MaherM.A.E. AutyY.H. RoosL.M. ZychowskiM.A. Fenelon Microstructure and lactose crystallization properties in spray dried nanoemulsions (2014), http://dx.doi.org/10.1016/j.foostr.2014.10.001 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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*Graphical Abstract
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Maher Pg. 1 Highlights
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x Powders prepared from nanoemulsions crystallized more quickly than those prepared from conventional emulsions x Polarised light microscopy was used to confirm onset of crystallization and was consistent with dynamic vapour absorption data. x CLSM and cryo-SEM showed the distribution of fat globules within the particles in situ. Cryo-SEM was particularly useful for characterising nano-sized fat droplets (<200 nm) which were below the resolution limit of confocal microscopy. x It is suggested that reduced protein in the continuous phase with lactose contributed to the observed increase in crystallization rate in powders prepared from nanoemulsions
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Maher Pg. 2 12
Title: Microstructure and lactose crystallization properties in spray dried nanoemulsions
13 14
P.G. Maher1, 2, M.A.E. Auty1*, Y.H. Roos2, L.M. Zychowski1, 2 M.A. Fenelon1,
15
1
16
Fermoy, Co. Cork, Ireland and 2Food Technology, School of Food and Nutritional Sciences,
17
University College Cork, Cork, Ireland.
cr
ip t
Food Chemistry & Technology Department, Teagasc Food Research Centre, Moorepark,
18 Author’s Affiliation and Address:
20
Patrick G. Maher, Research Student, Food Chemistry & Technology Department, Teagasc
21
Food Research Centre, Moorepark, Cork, Ireland.
22
Mark A. Fenelon, Principal Research Officer, Food Chemistry & Technology Department,
23
Teagasc Food Research Centre, Moorepark, Cork, Ireland.
24
Yrjö H. Roos, Professor, School of Food and Nutritional Sciences, University College Cork,
25
Cork, Ireland.
26
Lisa M. Zychowski, Research Student, Food Chemistry & Technology Department, Teagasc
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Food Research Centre, Moorepark, Cork, Ireland.
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Mark A.E. Auty, Senior Research Officer, Food Chemistry & Technology Department,
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Teagasc Food Research Centre, Moorepark, Cork, Ireland.
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*Contact information for Corresponding Author:
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Dr. Mark Auty, Food Chemistry & Technology Department, Teagasc Food Research Centre,
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Moorepark, Fermoy, Co. Cork, Ireland. Tel: +3532542442.
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email: [email protected].
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Maher Pg. 3 Abstract
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The objective of this study was to characterize lactose crystallization behaviour and
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microstructure of spray dried nanoemulsions with different fat globule sizes (FGS). Powders
40
of the same composition (57.7% w/w lactose, 12.3% w/w sodium caseinate, 27.7% w/w
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sunflower oil, and 2.3% w/w water) but different FGS (mean diameters 1100 nm and 160
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nm) prior to spray drying were manufactured. Differences in lactose crystallization were
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studied using dynamic vapour sorption (DVS) and polarized light microscopy (PLM).
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Crystallization kinetics was modelled using the Avrami and Yang equations. Results showed
45
that lactose crystallized in three dimensions and more rapidly in powders with a smaller FGS.
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PLM images showed a higher rate of crystal formation for smaller FGS powders when stored
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for up to 4 days at 55% relative humidity. Confocal laser scanning microscopy (CLSM) and
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cryo-scanning electron microscopy (Cryo-SEM) images indicated the more evenly distributed
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small fat globules inside powder particles prepared from spray-dried nanoemulsions. The
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surface of powder particles was uneven and ruptured post lactose crystallization. Crystals of
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presumptive anhydrous D- and E-lactose (5:3 molar ratio) appeared after humidification.
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Results showed powder particles of the same composition were altered in lactose
53
crystallization characteristics by changing the FGS of emulsions pre spray drying.
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Keywords: Lactose crystallization, dynamic vapour sorption, polarized light microscopy,
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confocal laser scanning microscopy, cryo-scanning electron microscopy.
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Maher Pg. 4 62
Highlights
63 x Reducing FGS of emulsions pre drying increased rate of lactose crystallization.
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x Crystallization occurred in three dimensions based on the Avrami exponent.
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x CLSM and cryo-SEM showed in situ smaller FGS in powders from nanoemulsions.
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x Needle-like anhydrous lactose crystals were observed by cryo-SEM and CLSM.
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x Particles were aggregated and lost their shape post lactose crystallization.
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Maher Pg. 5 87
1. Introduction
88 In dairy products, the degree of lactose crystallization has a significant effect on ingredient
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and food properties, such as texture and flavour. Crystallization of lactose in milk powders
91
results in lumping and caking which has a negative impact on powder reconstitution (Lai and
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Schmidt, 1990). Component crystallization also causes the release of entrapped lipids in
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powders (Fäldt and Bergenståhl, 1996; Shimada et al., 1991) making them more susceptible
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to oxidation.
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Various methods can be used to quantify crystallization of amorphous sugars. Isothermal
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differential scanning calorimetry (DSC) was used by Roos and Karel (1990) and Kedward et
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al. (1998) to determine the crystallization of freeze dried lactose and sucrose at low water
99
contents (< 5%) and high temperatures (365 – 400 K). X-ray diffraction (XRD) was used by
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Jouppila et al. (1997) and Miao and Roos (2005) to monitor crystallization kinetics of lactose,
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trehalose and lactose/trehalose mixtures. Mahlin et al. (2004) used atomic force microscopy
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(AFM) to quantify crystallization.
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Lactose crystallization can be measured by gravimetric means, by monitoring changes in
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mass of powder samples upon humidification (Iglesias and Chirife, 1978; Jouppila and Roos,
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1994; Lai and Schmidt, 1990). Crystallization results in sorbed water release, which is
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measured from mass decrease in a humidified powder (Burnett et al., 2004; Burnett et al.,
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2006). This method is most successfully used at ambient temperatures where the
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crystallization rate is quite slow and can be easily monitored (Kedward et al., 2000).
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Maher Pg. 6 Crystallization kinetics can be modelled using various relationships. The Avrami equation
112
(Avrami, 1939, 1940) has been used by several researchers (Arvanitoyannis and Blanshard,
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1994; Haque and Roos, 2005; Kedward et al., 2000; Mazzobre et al., 2003). Derivative
114
models from the Avrami equation include the Yang equation (Yang et al., 2010) and
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Urbanovici-Segal equation (Urbanovivi and Segal, 1990). The Urbanovici-Segal equation
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includes a parameter r (r > 0) that determines how far the model deviates from the Avrami
117
equation (Soutari et al., 2012). Other kinetic models used to determine lactose crystallization
118
are the Williams-Landel and Ferry (WLF) equation (Roos and Karel, 1991) and the Hoffman
119
equation (Arvanitoyannis and Blanshard, 1994). The Arrhenius equation can be used to
120
determine the activation energy (EA) required for crystallization (Schmidt et al., 1999). The
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Avrami equation determines the rate constant (k) and exponent (n) that, respectively,
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determine how fast the material crystallizes and in how many dimensions. Powders stored at
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increasing temperatures and humidities crystallize at faster rates (Soutari et al., 2012).
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Polarized light microscopy (PLM) is a useful technique in distinguishing crystalline from
125
non-crystalline material (Hartel, 2001) and can be used to directly measure the rate of crystal
126
growth. Mazzobre at al. (2003) used polarized light videomicroscopy (PLV), in conjunction
127
with DSC, to determine the isothermal crystallization kinetics of lactose and lactose-trehalose
128
mixtures. The PLV method proved a useful way of directly observing individual crystal
129
growth and morphological aspects that were undetectable by DSC.
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The distribution of fat globules has a significant effect on functional properties of dairy
132
powders (Pisecky, 1997). Techniques such as CLSM and cryo-SEM are useful in highlighting
133
microstructural differences which help explain why dairy powders have various functional
134
properties. CLSM is widely used for studying the microstructure of cheese, milk powder and
135
chocolate (Auty et al., 2001). McKenna (1997) examined whole milk powder using CLSM.
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Maher Pg. 7 Kelly et al. (2014) used CLSM to analyse the distribution of fat droplets in powder samples
137
where different fat blends were stabilized by sodium caseinate. Samples were dual-labelled
138
with Nile Red / Fast Green FCF to show the distribution of fat and protein, respectively,
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throughout the powder particles. Air vacuoles were also visible in powders in images
140
produced by CLSM. SEM techniques have been developed to examine the outer and inner
141
structures of foods (Soottitantawat et al., 2003). SEM has been used in studies of
142
microencapsulation properties of powder structures stabilized by sodium caseinate (Fäldt and
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Bergenståhl, 1996; Hogan et al., 2001a, b). Cryo-SEM is a useful technique in analyzing
144
nano-sized particles embedded in food matrices (Dudkiewicz et al., 2011).
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The objectives of the present study were (1) to investigate the effects of reducing the FGS of
147
emulsions prior to spray drying on water-induced lactose crystallization during storage; and
148
(2) to characterize powder microstructure using correlative microscopy techniques (PLM,
149
CLSM and cryo-SEM) to help explain differences of the physical properties of the powders.
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2. Materials and Methods
152
2.1 Materials
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Two powders, containing 57.7% w/w lactose, 12.3% w/w sodium caseinate and 27.7% w/w
154
sunflower oil, were used (Maher et al., (2014). The powders differed in their mean FGS as
155
they were both homogenized (60 °C with a single pass) at different pressures prior to spray
156
drying. Conventional emulsions were produced using in-line two-stage homogenization
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(model NS20006H, GEA Niro, Soavi, Parma, Italy) at first- and second-stage pressures of
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13.79 MPa and 3.45 MPa, respectively, giving a FGS of ~ 1100 nm. Nanoemulsions were
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produced by microfluidization with a Microfluidizer (model M-110EH, Microfluidics,
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Newton, MA, USA) at 100 MPa, giving a FGS of ~ 155 nm. All emulsions were spray dried
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Page 8 of 33
Maher Pg. 8 at the same conditions (inlet/outlet dryer temperatures of 180 °C/80 °C) with a single stage
162
pilot-scale spray dryer (Anhydro F1 Lab, Copenhagen, Denmark) equipped with a two-fluid
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nozzle atomization system (Type 1/8 JAC 316ss) and counter-flow drying. Resulting powders
164
were labelled C and N for conventional emulsion powders (FGS ~ 1100 nm) and
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nanoemulsions powders (155 nm), respectively.
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168
2.2.1. Dynamic vapour sorption
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Water sorption isotherms of powders were obtained from the DVS Data Analysis Suite in
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Microsoft Excel using a DVS-1 analyser (Surface Measurement Systems, London, UK)
171
equipped with a Cahn microbalance. Powder samples (~ 30 mg) were placed in the sample
172
pan with an empty pan used as a reference. Samples were humidified at 55% relative
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humidity (RH) on the DVS for two days (48 h) for C and N at 25ºC. Mixing dry nitrogen gas
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(200 mL/min) with saturated water vapour in the correct proportion using mass flow
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controllers was used to get accurate RH readings. Samples were measured from three
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replicate spray drying trials.
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2.2.2. Polarized light microscopy
179
Samples were examined using an Olympus BX51 light microscope (Olympus BX-51,
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Olympus Corporation, Tokyo, Japan) with a 10x dry objective lens using polarized light.
181
Digital images (TIFF, 8-bit) were taken using a Jenoptik C14 Imagic camera, and captured.
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Powders were analysed pre- and post- lactose crystallization following storage over a
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saturated solution of Mg(NO3)2 (55% RH) for 4 days at 20 °C. Crystalline regions appeared
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as bright areas on the micrographs.
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Page 9 of 33
Maher Pg. 9 2.2.3. Confocal laser scanning microscopy
187
Visualization of powder particles was carried out using a Leica TCS SP5 confocal laser
188
scanning microscope (CLSM; Leica Microsystems CMS GmbH, Wetzlar, Germany). Powder
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samples were placed onto a glass slide and labelled using a mixture of Fast Green and Nile
190
Red or Nile Red alone (Auty et al., 2001). The dye mixture comprised Fast Green (aq. 0.01 g
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/ 0.1 L) and Nile Red dissolved in polyethylene glycol mol. wt. 400 (0.1g / 0.1 L) mixed in a
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ratio that both allowed diffusion of the dye molecules into the powder particles whilst
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maintaining particle integrity and preventing solubilisation. Based on trial and error, pre- and
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post-lactose crystallized powders were labelled, respectively, with 1:40 or 1:20 ratios of Fast
195
Green to Nile Red. Dual excitation using 488nm/633nm was utilized for all Nile Red and Fast
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Green mixtures, while single excitation at 488 nm was used for the Nile Red images.
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Representative images of each sample were taken using 63x oil immersion objective with
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numerical aperture 1.4. Z-stacks were obtained in order to generate a three-dimensional
199
structure of the particle and to identify surface fat staining. Red and, Green pseudo-coloured
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images (8-bit), 512 x 512 pixels in size, were acquired using a zoom factor of 1. A minimum
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of four z-stacks were taken per sample, with representative micrographs being shown.
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Samples were measured pre and post lactose crystallization (55% RH for 3 days).
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2.2.4. Cryo-scanning electron microscopy (Cryo-SEM)
205
Cryo-SEM was used to examine the microstructure of powders. Powders were sprinkled onto
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the surface of OCT embedding compound (Agar Scientific Ltd, UK) on a slotted aluminium
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stub, then plunge-frozen into liquid nitrogen slush at
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to the cold stage using the Alto 2500 cryo-transfer device (Gatan Ltd., Oxford, UK). A cold
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scalpel was used to fracture samples. The sample was cooled to
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at
Page 10 of 33
Maher Pg. 10 imaging at !"#$ '=?Q
[
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Zeiss SMT Ltd., Cambridge, UK), operating at 2 kV and a working distance of 6 mm, was
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used to obtain secondary electron images. Representative micrographs were taken in order to
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visualize surface characteristics, including surface fat and lactose crystals, where present.
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Samples were analyzed pre- and post- lactose crystallization (55% RH for 3 days).
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218
Spray drying experiments were carried out in triplicate. One-way analysis of variance
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(ANOVA) was used with Minitab 15 (Minitab Ltd., Coventry, UK) to determine significant
220
differences between lactose crystallization kinetics of both sets of powders by Fisher’s one-
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way multiple comparison test. Results were deemed statistically significant if P < 0.05.
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222 3. Results and discussion
224
3.1 Water sorption kinetics
225
Water sorption kinetics of powders C and N were measured by DVS at 55% RH and 25 °C.
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These conditions were chosen since Burnett et al. (2004) showed that below 30% RH lactose
227
crystallization will not occur and above 58% RH lactose crystallization is nearly
228
instantaneous.
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Figure 1a shows the mean water uptake in powders from 3 replicate samples on a solids-non-
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fat (SNF) basis over a 2880 min (48 h) period. It is shown that there was no difference in
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initial water sorption behaviour of the powders. The mean maximum sorption rate of water in
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the first 60 min was 7.4 g water / 100 g SNF for C, and 7.9 g water / 100 g SNF for N.
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Maximum water sorption occurred in approximately 140 min, upon which there was a
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decrease in mass and therefore evidence for onset of lactose crystallization (Burnett et al.,
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Maher Pg. 11 2004; Burnett et al., 2006). Powders C and N shown in Figure 1a crystallized at different
237
rates, with lactose crystallising faster in N than in C. The mass of water decreased until full
238
crystallization was achieved towards the end of the run. The mean crystallization rate of
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lactose in each powder showed 0.9 g water / 100 g SNF lost per hour for C and 1.9 g water /
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100 g SNF lost per hour for N (measured between 700 and 760 min). The onset (m0) and end
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points (m\) of lactose crystallization were found by calculating the intersection point of
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tangent lines to the respective baselines using the experimental data in Figure 1a. The mean
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onset points for C and N were 483 min and 424 min, respectively, showing that N started to
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crystallize before C. N also had a mean end point of 1098 min compared to a mean end point
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of 1240 min for C. This clearly showed that lactose crystallized faster in powders from
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nanoemulsions (674 min in total) than in powders from conventional emulsions (757 min in
247
total). This result was similar to that of Maher at al. (2014) who found using Differential
248
Scanning Calorimetry (DSC) that lactose in powders from nanoemulsions crystallized at
249
significantly (P < 0.05) lower temperatures than in powders from conventional emulsions.
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Maher at al. (2014) explained that this was due to nanoemulsions having a smaller FGS
251
which means that there was more surface area for protein to adsorb to, leaving less protein in
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the continuous lactose phase.
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More detailed quantitative analysis of lactose crystallization was achieved by converting the
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experimental data to the fraction crystallized over time, and subsequent results were analyzed
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statistically. This was calculated by comparing the mass of water absorbed by the powder at a
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given time (mt) to the mass of absorbed water at the end of crystallization (m\) and the mass
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of water absorbed at the onset of crystallization (m0). Then:
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Crystallization T t
m0 mt m0 mf
(1)
Page 12 of 33
Maher Pg. 12 The resulting isotherm is shown in Figure 1b and is the mean of 3 replicate powders of each
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type (C and N). The time has been normalized so that the onset of lactose crystallization is
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time zero and the y-axis has been normalized to reflect the crystallization fraction. Before
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lactose crystallization, the crystalline fraction was assumed to be zero and it was assumed to
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be one after crystallization. Various different equations have been used in literature to model
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crystallization kinetics, the most common of which is the Avrami model (1939):
266
Tt
267
where k is the rate constant (min-1) and n is the Avrami exponent. The rate constant (k)
268
quantifies how fast the crystals grow indicating the stability of the system (Gaisford et al.,
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2009) and the Avrami exponent (n) defines the mechanism of crystallization. The Avrami
270
rate constant mainly depends on the crystallization temperature (Kawamura, 1979) and
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typically follows an Arrhenius-type temperature dependency. It takes both nucleation and
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crystal growth into account (Sharples, 1966). An Avrami exponent value of 1 represents
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nucleation and crystal growth from surfaces, a value of 2 represents crystal growth from
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edges, and a value of 3 indicates crystal growth in three dimensions. Non-integer n values can
275
also be obtained and they represent mixed crystallization mechanisms (Cahn, 1956; Soutari et
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al., 2012). The sigmoidal shape of the curve in Figure 1b is typical of those produced using
277
the Avrami equation. The larger the Avrami exponent (n) the more sigmoidal the
278
crystallization curve (Avrami, 1940). Transformation rates are low at the beginning and end
279
of the transformation, but rapid in between. Initially there is a slow rate due to the time
280
required to form a significant number of nuclei of the new phase. The nuclei grow rapidly
281
into particles, during the intermediate phase, and consume the old phase while nuclei
282
continue to form in the remaining parent phase. When the transformation is nearly complete,
283
there is less material to transform to nuclei and the production of new particles becomes slow.
284
Also, the already existing particles touch each other forming a boundary where growth stops.
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(2)
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Maher Pg. 13 This is not accounted for in the Avrami model as it assumes a constant crystal growth rate,
286
which is why the model breaks down as complete crystallization approaches (Soutari et al.,
287
2012).
288
The experimental data was also modelled using a modified version of the Avrami equation
289
from Yang et al. (2010):
290
Tt
291
The same equation was derived by Tobin et al. (1974). Crystallization isotherms were fitted
292
to Eqs. (2) and (3) and the results were compared to the experimental data in Table 1. The
293
Avrami model was found to better describe the experimental crystallization process than the
294
Yang model. The mean Avrami rate constants of 1.1 x 10-8 min-1 (C) and 1.2 x 10-7 min-1 (N)
295
are significantly different (P < 0.05) showing that lactose in powders from nanoemulsions
296
crystallized faster than in conventional emulsions (Table 1). The Avrami integers are very
297
close to 3 indicating the crystals are growing in three dimensions (Soutari et al., 2012).
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Half-times of lactose crystallization are useful in predicting the storage stability of lactose
299
containing products (Jouppila et al., 1997). The crystallization half-time is calculated using
300
the rate constant (k) and exponent (n) in the following equation:
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(3)
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1/ n
§ ln 2 · ¨ ¸ © k ¹
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t1/ 2
302
The t1/2 values, calculated using the Avrami k and n values, were very similar to those found
303
experimentally (Table 1). High coefficients of determination (r2) and similar t1/2 values
304
indicate that the Avrami equation models the experimental data to a high degree of accuracy.
305
Figure 1b shows that Avrami crystallization values are very similar to experimental values.
306
Values found using the Yang equation were less accurate than those from the Avrami
307
equation (Table 1). The coefficient of determination was lower and the t1/2 was much
308
different to the experimental value. The exponents (n) of 4.39 (C) and 4.31 (N) were greater
(4)
Page 14 of 33
Maher Pg. 14 and rate constants (k) were lower than Avrami constants. Similar results were reported by Al-
310
Mulla (2007) who found that the Yang (or Tobin) model did not fit as well as the Avrami
311
model because it gave higher n values and lower r2 values for the crystallization of polymers.
312
The rate constants (k) obtained in this study (1.1 – 12 x 10-8 min-1) were much lower than
313
those obtained by Schmidt et al. (1999), who measured the crystallization kinetics of
314
amorphous lactose at 25 °C in the range 52.5 – 57.5% RH (5.6 – 15.5 x 10-3 min-1). Jouppila
315
et al. (1997) found that the Avrami model did not work for skimmed milk powder (SMP) at
316
53.8% RH, but did work at humidities ]^^!_!` ${? [!
317
x 10-10 min-1) with a corresponding increase in crystallization half-time (40 h) compared to
318
the powders in this study, even at the higher RH of 66.2%. Similar Avrami indexes were
319
obtained for SMP (~ 3), indicating crystal growth in three dimensions. Overall, this shows
320
that protein had a significant effect on lactose crystallization kinetics. When protein was not
321
present, lactose crystallized quickly (Schmidt et al., 1999). The 11% sodium caseinate in this
322
study caused delayed lactose crystallization, and increasing the protein content to 35% (for
323
SMP) further delayed lactose crystallization (Jouppila et al., 1997). It is postulated that
324
nanoemulsions have less non-adsorbed protein than conventional emulsions due to their
325
smaller FGS giving increased surface area for protein adsorption. Therefore, this study
326
suggests that not only is the total amount of protein important, but the amount of non-
327
adsorbed protein that has the most significant effect on lactose crystallization.
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To the author’s best knowledge no other studies have compared crystallization of lactose in
329
powders with significantly different FGS. The best comparison can be made with McCarthy
330
et al. (2013) who showed that lactose crystallization occurred at lower ware contents with
331
decreasing protein content (i.e., less protein in the continuous phase) for infant formula
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powders. Similar results were found by Thomas et al. (2004) who found that increasing E-
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Maher Pg. 15 333
lactoglobulin contents increased the humidity at which lactose crystallized in E-
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lactoglobulin/lactose mixtures.
335 3.2 Microscopy
337
3.2.1 Polarized light microscopy
338
Figure 2 shows PLM images of powders C and N when stored in a desiccator over a saturated
339
salt solution of Mg(NO3)2 at 55% RH for 0, 1, 2, 3, and 4 days. The micrographs are
340
consistent with results obtained from DVS, showing that lactose crystals formed more
341
quickly in powder N than powder C over 4 days. Both powders at day 0 showed no evidence
342
of lactose crystals. This was in contrast to other studies (Haque and Roos, 2005; McCarthy et
343
al., 2013; Roetman, 1979) that showed lactose crystals present in fresh powders. At day 1, no
344
crystals were seen in powder C but some small isolated crystals were seen in powder N
345
(Figure 2d). At day 2, some crystals had formed in powder C (Figure 2e) with increased
346
growth of crystals on the edges of powder particles in powder N (Figure 2f). Surface crystals
347
are evident in powder C at day 3 (Figure 2g) but there are noticeably more crystals present in
348
powder N (Figure 2h). By day 4, both powders C and N showed crystallization throughout
349
the particles.
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3.2.2. Confocal laser scanning microscopy
352
Confocal laser scanning microscopy results are shown in Figure 3. Labelling with Nile
353
Red/Fast Green highlighted the fat and protein phases of the powder and showed air vacuoles
354
as dark regions within the particle (Figure 3a, b, arrows). Discrete spherical fat droplets, most
355
of which are 1 - 5 Pm in diameter, were clearly seen within powder C (Figure 3a). By
356
contrast, fat droplets in Powder N were small (Figure 3b); although some individual droplets
357
(~ 0.2 - 1Pm diameter) were visible, there was considerable co-localization of the Nile Red
Page 16 of 33
Maher Pg. 16 and Fast Green signals indicating that most fat droplets were below the resolution limit of the
359
CLSM. The protein phase of both powders is shown in red. The protein is more clearly
360
observed in powder C than powder N due to the fat globules being less numerous and evenly
361
spaced, allowing more gaps for the protein phase to be visualized. Very little surface fat was
362
visible for either powder (Figure 3a, b), in contrast with some agglomerated whole milk
363
powder particles that have shown evidence of surface fat pooling (McKenna, 1997). No
364
evidence of lactose crystals was found in fresh powders (Figure 3a, b).
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Powders stored after 4 days at 55% RH are shown in Figure 3 c, d, e, f. Particles from both
367
powders appeared to have deformed and were more agglomerated than fresh powders. Figure
368
3c shows large free fat droplets (arrows) > 5 Pm in diameter as well as fat droplets and pools
369
within the particles. Powder N shows generally less free fat but had more obvious elongated
370
or needle-shaped crystals seen by negative contrast (Figure 3d, arrows). The protein signal
371
was fairly weak and appeared localized at the surface and outer edges of the particles (Figure
372
3 c, d). This may be due to high level of lactose crystallization preventing the stain from
373
diffusing into the particle core.
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To study the fat phase in more detail and reduce possible staining-induced coalescence,
376
powders were stained with Nile Red solubilized in polyethylene glycol, mol. wt. 400 and
377
results are shown in Figure 3e, f. The fat globules within the particles no longer appear
378
spherical as in fresh powders, probably due to lactose crystallizing around them and forcing
379
them into irregular pools (see inset Figure 3e). Powder N appeared to have less free fat and
380
more intact air vacuoles, indicating that powder particles from nanoemulsions are more stable
381
to crystallization-induced fat rupture.
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Page 17 of 33
Maher Pg. 17 Few studies have looked at powders using CLSM post lactose crystallization. McCarthy et al.
384
(2013) used CLSM and found free fat partially covering lactose crystals post crystallization
385
of a model infant formula after it had leaked from the interior of the powder. McKenna
386
(1997) showed that for whole milk powder, when stained for fat only, that the entire powder
387
particle could be seen with lactose crystals clearly evident from negative contrast. The lactose
388
crystals in that study were rhomboidal D-crystals due to the lactose crystallizing in the milk
389
prior to spray drying. Needle-shaped crystals were seen post lactose crystallization in the
390
current study. Jouppila et al. (1997) and Bushill et al. (1965) reported that lactose in skimmed
391
milk powder crystallized at 55% RH in a 5:3 molar ratio of anhydrous D-lactose: E-lactose,
392
therefore our results are assumed to be the same.
an
us
cr
ip t
383
M
393 3.2.3 Cryo-scanning electron microscopy
395
It is important to characterize the outer topography of particles as it can give information on
396
flow properties of spray dried powders (Soottitantawat et al., 2003). Cryo-SEM images are
397
presented in Figures 4 and 5. The surface morphology of fresh powders from C and N
398
powders is shown in Figures 4a and b, respectively. The surfaces of all powders appeared to
399
be very smooth with little indentation and powder rupture, similar to data of Hogan et al
400
(2001b) who also used sodium caseinate as an emulsifier. Sodium caseinate is a very efficient
401
emulsifier and dominates the powder surface, even in small concentrations, due to a higher
402
surface activity of protein in water during the drying process (Fäldt and Bergenståhl, 1996).
403
Very little solvent extractable free fat was measured previously by Maher et al. (2014), with
404
powder N (0.4%) having a significantly lower FF content than powder C (2.3%). There was
405
little evidence of free fat on the surface of these powders. Fractured particles are shown in
406
Figure 4c, d, e, f. The advantage of cryo-SEM is that the air vacuoles are easily visible and
407
well preserved. Both powders showed high levels of occluded air in the form of rounded
Ac ce p
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d
394
Page 18 of 33
Maher Pg. 18 408
vacuoles (Figure 4c, d, arrows), although powder N appeared to have slightly larger vacuoles
409
and thinner cross-walls than powder N. Vacuoles were distributed evenly throughout the
410
powder particles and were mostly in the size range 5 – 50 Pm.
ip t
411 Conventional emulsion powders (C) have an uneven distribution of oil droplets spread
413
throughout the powder particles, whereas nanoemulsion powders (N) have an even
414
distribution of much smaller oil droplets. The size of the oil droplets was ~ 1100 nm for C
415
and ~ 150 nm for N. The smaller oil droplets in nanoemulsions were more embedded in the
416
matrix and therefore less likely to be extracted upon addition of solvent. No evidence of
417
lactose crystals was found in any of the fresh powders. Higher magnifications revealed the
418
powder wall structure at the nano-scale (Figure 4e, f, g, h). In powder C spherical fat
419
droplets, mostly 200 nm to 5 Pm in diameter, are seen embedded within the wall matrix
420
(Figure 4 e, f, arrows). Fat droplets were smaller in powder N (< 200 nm diameter) and could
421
only be seen at very high magnifications (Figure 4h, arrow).
us
an
M
d
te
422
cr
412
The powders were also examined by cryo-SEM post lactose crystallization (Figure 5). At low
424
magnification, agglomeration (or melding) of powder particles was evident (Figure 5a, b).
425
Higher magnification revealed the presence of radiating clusters of needle-like crystals on
426
powder surfaces (Figure 5c, d). Surfaces of lactose-crystallized powders were more uneven
427
with various indentations and holes. The irregular surface, as a result of lactose
428
crystallization, was also observed by Murrieta-Pazos et al. (2011). Faldt and Bergenståhl
429
(1996) found using electron spectroscopy for chemical analysis (ESCA) that a protein film
430
dominates the powder surface even after lactose crystallization. Solid bridges formed
431
between particles after lactose crystallization making them agglomerate and cluster, which is
432
in contrast to the more discrete individual particles from the fresh powder. No large crystals
Ac ce p
423
Page 19 of 33
Maher Pg. 19 were observed on the powder surface. Faldt and Bergenståhl (1996) postulated that fat
434
globules reduce the size of lactose crystals formed after humidification. Fractured particles
435
showed crystals embedded within the wall and also projecting into vacuoles (Figure 5e, f,
436
arrows). There was little evidence of intact spherical fat droplets in either powder, suggesting
437
that they had been disrupted by lactose crystals. This observation is consistent with CLSM
438
results which showed irregular fat pools dispersed within the powder particles (Figure 3e)
439
and highlights the advantage of a correlative microscopy approach. Cryo-SEM had the
440
advantages of good structural preservation and high resolution but could not easily
441
distinguish between fat, protein or amorphous lactose. CLSM revealed the distribution of fat
442
in powders more clearly, while PLM revealed the onset and progress of crystallization over
443
time and confirmed DVS data which indicated that powders prepared from nanoemulsions
444
crystallized at a faster rate. Combining cryo-SEM with CLSM and PLM thus provided a
445
powerful set of tools for characterising the microstructures of dairy powders.
446
te
d
M
an
us
cr
ip t
433
Conclusions
448
Powders prepared from nanoemulsions crystallized significantly (P < 0.05) more quickly
449
than those prepared from conventional emulsions, as measured using DVS. Rates were
450
calculated with the Avrami equation and crystals were found to form in three dimensions.
451
Evidence of crystal growth was observed with PLM and showed the same trend as results
452
from DVS. CLSM and cryo-SEM show differences in powder structure with powders from
453
nanoemulsions having smaller fat globules that are evenly distributed throughout powder
454
particles. It is suggested that reduced protein in the continuous phase with lactose contributed
455
to observed increase in crystallization rate in powders prepared from nanoemulsions.
456
Elongated lactose crystals (probably a mixture 5:3 molar ratio of D and E-lactose), initially
457
at the surface but progressively occurring within the particles, were observed post
Ac ce p
447
Page 20 of 33
Maher Pg. 20 humidification at 55% RH. To the authors’ knowledge, there have been no studies on
459
powders prepared from nanoemulsions using CLSM, SEM, or cryo-SEM that show the
460
distribution of fat globules within the particles in situ. This paper provides a useful insight
461
into how reducing the FGS of emulsions pre spray drying impacts upon the physical
462
properties of powders.
ip t
458
cr
463 Acknowledgements
465
The authors would like to acknowledge the National Food Imaging Centre, Teagasc Food
466
Research Centre, Moorepark, Fermoy, Co. Cork, Ireland for use of the microscopes. The
467
project was funded by the Irish Department of Agriculture, Food and the Marine under the
468
Food Institutional Research Measure (FIRM).
M
469
479
d
Industrial relevance
te
Relatively little is known about the physical and chemical properties of nano-sized emulsions when spray dried. Drying of milk-based food ingredients is a major industry and high-shear processing is increasingly used to reduce fat droplet size to improve stability. This paper highlights one unintended consequence of reducing fat droplet size, namely increasing the tendency for lactose to crystallize. This should be factored in to any future formulations that involve high shear processing of milk derived ingredient s for subsequent spray drying.
Ac ce p
470 471 472 473 474 475 476 477 478
an
us
464
Page 21 of 33
Maher Pg. 21 479 480
Table 1 Summary of kinetic parameters obtained by fitting the crystallization isotherms to the
481
Avrami model and Yang model.
ip t
482 483
n
t1/2
r2
k
(min-1)
-
(min)
-
(min-1)
C
1.1 x 10-8 a
3.06 a
363 a
0.995
7.3 x 10-12 a
N
1.2 x 10-7 b
2.76 b
287 b
0.985
2.3 x 10-9 a
485
a-b
486
way ANOVA.
490 491 492 493 494
r2
t1/2
-
(min)
-
(min)
4.39 a
323 a
0.976
368 a
4.31 a
262 b
0.970
285 b
d
M
Values within a column not sharing a common letter differ significantly (P < 0.05) by one-
te
489
t1/2
Ac ce p
488
Experimental
n
an
k
Powder
487
Yang
us
Avrami
cr
484
495 496 497 498 499
Page 22 of 33
Maher Pg. 22 500 Figure captions
502
Figure 1. a) Dynamic vapour sorption graph of control (C) and nanoemulsion (N) powders
503
held at 55% RH for 24 h showing lactose crystallization. b) Crystallization isotherms for C
504
and N at 25 °C for experimental data and those calculated using the Avrami equation.
506
Figure 2. Polarized light (crossed polars) micrographs of control (a,c,e,g,i) and nanoemulsion
507
(b,d,f,h,j) powders. after storage for 0 (a,b), 1 (c,d), 2 (e,f), 3 (g,h) and 4 (i,j) days at 55%
508
relative humidity. Arrows indicate small crystals, scale bar = 100 Pm.
an
us
cr
505
ip t
501
509
Figure 3. Confocal scanning laser micrographs of showing internal microstructures of control
511
(a,c,e) and nanoemulsion (b,d,f) powders after storage for 0 (a,b) and 4 (c,d,e,f) days at 55%
512
relative humidity. Figure 4 a – d are fluorescently labelled with Nile red/fast green to show
513
fat (green) and protein (red); Figure 4 e & f are labelled with Nile Red to show fat phase.
514
Arrows indicate air vacuoles (a, b), discrete fat droplets (c) and solid lactose crystals (d). Fig
515
3e inset shows irregularly shaped coalesced fat regions. Scale bars = 100 Pm.
d
te
Ac ce p
516
M
510
517
Figure 4. Cryo-scanning electron micrographs of control (a,c,e,g) and nanoemulsion (b,d,f,h)
518
powders prior to crystallization. Images show powder particle surface morphology (a,b) and
519
interior features of fractured particles (c – h). Arrows indicate air vacuoles (c,d) and fat
520
droplets (e,g,h). Scale bars = 20 Pm (a,b), 10 Pm (c,d), 2 Pm (e,f) and 500 nm (g,h).
521 522
Figure 5. Cryo-scanning electron micrographs of control (a,c,e) and nanoemulsion (b,d,f)
523
powders after storage for 4 days at 55% relative humidity showing crystals (arrowed) on the
Page 23 of 33
Maher Pg. 23 powder surfaces (a,b,c,d) and also interior of fractured particles. Scale bars = 20 Pm (a,b), 5
525
Air vacuole Pm (c,d) and 2 Pm (e,f).
526
Figures
Ac ce p
te
d
M
an
us
cr
ip t
524
527 528
Figure 1.
529 530
Page 24 of 33
Maher Pg. 24 531 532 533
a
b
c
d
e
f
ip t
534 535
cr
536
538
us
537
an
539 540
te
544 545 546 547 548
g
549 550 551 552
i
Ac ce p
543
d
542
M
541
h
j
553 554 555
Page 25 of 33
Maher Pg. 25 556
558 559
Figure 2.
a
b
c
d
ip t
557
560
cr
561 562
us
563
an
564
566
M
565
567
d
568
te
569
571 572 573 574 575 576
Ac ce p
570
e
f
577 578 579 580
Page 26 of 33
Maher Pg. 26 581 582 583
Figure 3.
a
b
c
d
ip t
584 585
cr
586
589
an
588
590
M
591 592
597 598 599 600 601
Ac ce p
595 596
f
te
e
d
593 594
us
587
g
h
602 603 604
Figure 4.
605
Page 27 of 33
Maher Pg. 27 606 607 608
a
b
c
d
ip t
609 610
cr
611 612
an
614 615
M
616 617
d
618
e
f
te
619
622 623 624 625 626
Ac ce p
620 621
us
613
Figure 5.
627 628 629 630
Page 28 of 33
Maher Pg. 28 631 632 References
634
Al-Mulla, A., (2007). Isothermal crystallization kinetics of poly(ethylene terephthalate) and
635
poly(methyl methacrylate) blends. eXPRESS Polymer Letters 1(6), 334-344.
636
Arvanitoyannis, I., Blanshard, J.M.V., (1994). Rates of Crystallization of Dried Lactose-
637
sucrose Mixtures. Journal Of Food Science 59(1), 197-205.
638
Auty, M.A.E., Twomey, M., Guinee, T.P., Mulvihill, D.M., (2001). Development and
639
application of confocal scanning laser microscopy methods for studying the distribution of fat
640
and protein in selected dairy products. Journal of Dairy Research 68, 417-427.
641
Avrami, M., (1939). Kinetics of phase change. I. General theory. Journal of Chemical Physics
642
7, 1103-1112.
643
Avrami, M., (1940). Kinetics of Phase Change II. Transformation-Time Relations for
644
Random Distribution of Nuclei. Ibid 8, 212-224.
645
Burnett, D., Thielmann, F., Booth, J., (2004). Determining the critical relative humidity for
646
moisture-induced phase transitions. International Journal of Pharmaceutics 287, 123-133.
647
Burnett, D., Thielmann, F., Sokoloski, T., Brum, J., (2006). Investigating the moisture-
648
induced crystallization kinetics of spray-dried lactose. International Journal of Pharmaceutics
649
313, 23-28.
650
Bushill, J.H., Wright, W.B., Fuller, C.H.F., Bell, A.V., (1965). The crystallisation of lactose
651
with particular reference to its occurrence in milk powder. Journal of the Science of Food and
652
Agriculture 16(10), 622-628.
653
Cahn, J.W., (1956). Transformation kinetics during continuous cooling. Acta Metallurgia 4,
654
572-575.
Ac ce p
te
d
M
an
us
cr
ip t
633
Page 29 of 33
Maher Pg. 29 Dudkiewicz, A., Tiede, K., Loeschner, K., Jensen, L.H.S., Wierzbicki, R., Boxall, A.B.A.,
656
Molhave, K., (2011). Characterization of nanomaterials in food by electron microscopy.
657
Trends in Analytical Chemistry 30(1), 28-43.
658
Fäldt, P., Bergenståhl, B., (1996). Changes in Surface Composition of Spray-Dried Food
659
Powders due to Lactose Crystallization. LWT - Food Sci. Technol. 29(5–6), 438-446.
660
Gaisford, S., Verma, A., Saunders, M., Royall, P.G., (2009). Monitoring crystallisation of
661
drugs from fast-dissolving oral films with isothermal calorimetry. International Journal of
662
Pharmaceutics 380, 105-111.
663
Haque, M.K., Roos, Y.H., (2005). Crystallization and X-ray diffraction of spray-dried and
664
freeze-dried amorphous lactose. Carbohydrate Research 340, 293-301.
665
Hartel, R., (2001). Measurements of crystalline structure in foods. In: Crystallization in
666
Foods. Aspen Publishers: Gaithersburg, MD, p 34-90.
667
Hogan, S.A., McNamee, B.F., O'Riordan, E.D., O'Sullivan, M., (2001a). Emulsification and
668
microencapsulation properties of sodium caseinate/carbohydrate blends. International Dairy
669
Journal 11(3), 137-144.
670
Hogan, S.A., McNamee, B.F., O'Riordan, E.D., O'Sullivan, M., (2001b). Microencapsulating
671
Properties of Sodium Caseinate. Journal of Agricultural and Food Chemistry 49, 1934-1938.
672
Iglesias, H.A., Chirife, I., (1978). Delayed crystallization of amorphous sucrose in humidified
673
freeze dried model systems. Journal of Food Technology 13, 137-144.
674
Jouppila, K., Kansikas, J., Roos, Y.H., (1997). Glass transition, water plasticization, and
675
lactose crystallization in skim milk powder. J Dairy Sci 80(12), 3152-3160.
676
Jouppila, K., Roos, Y.H., (1994). Glass transitions and crystallization in milk powders. J
677
Dairy Sci 77(10), 2907-2915.
678
Kawamura, K., (1979). The DSC Thermal Analysis of Crystallization Behaviour in Palm Oil.
679
Ibid 56, 753-758.
Ac ce p
te
d
M
an
us
cr
ip t
655
Page 30 of 33
Maher Pg. 30 Kedward, C.J., MacNaughtan, W., Blanshard, J.M.V., Mitchell, J.R., (1998). Crystallization
681
Kinetics of Lactose and Sucrose On Isothermal Differential Scanning Calorimetry. Journal Of
682
Food Science 63(2), 192-197.
683
Kedward, C.J., MacNaughtan, W., Mitchell, J.R., (2000). Isothermal and non-isothermal
684
crystallization in amorphous sucrose and lactose at low moisture contents. Carbohydrate
685
Research 329(2), 423-430.
686
Kelly, G.M., O’Mahony, J.A., Kelly, A.L., O’Callaghan, D.J., (2014). Physical
687
characteristics of spray-dried dairy powders containing different vegetable oils. Journal of
688
Food Engineering 122(0), 122-129.
689
Lai, H., Schmidt, S., (1990). Lactose crystallisation in skim milk powder observed by
690
hydrodynamic equilibria, scanning electron micrroscopy and 2H nuclear magnetic resonance.
691
J Food Sci 55(4), 994-999.
692
Maher, P.G., Roos, Y.H., Fenelon, M.A., (2014). Physicochemical properties of spray dried
693
nanoemulsions with varying final water and sugar contents. J. Food Eng. 126(0), 113-119.
694
Mahlin, D., Berggren, J., Alderborn, G., Engstrom, S., (2004). Moisture-Induced Surface
695
Crystallization of Spray-Dried Amorphous Lactose Particles Studied by Atomic Force
696
Microscopy. Journal of Pharmaceutical Sciences 93(1), 29-37.
697
Mazzobre, M.a.F., Aguilera, J.M., Buera, M.a.P., (2003). Microscopy and calorimetry as
698
complementary techniques to analyze sugar crystallization from amorphous systems.
699
Carbohydrate Research 338(6), 541-548.
700
McCarthy, N.A., Gee, V.L., Hickey, D.K., Kelly, A.L., O'Mahony, J.A., Fenelon, M.A.,
701
(2013). Effect of protein content on the physical stability and microstructure of a model
702
infant formula. International Dairy Journal 29(1), 53-59.
703
McKenna, A.B., (1997). Examination of whole milk powder by confocal laser scanning
704
microscopy. Journal of Dairy Research 64, 423-432.
Ac ce p
te
d
M
an
us
cr
ip t
680
Page 31 of 33
Maher Pg. 31 Miao, S., Roos, Y.H., (2005). Crystallization Kinetics and X-ray Diffraction of Crystals
706
Formed in Amorphous Lactose, Trehalose, and Lactose/Trehalose Mixtures. Journal Of Food
707
Science 70(5), E350-E358.
708
Murrieta-Pazos, I., Gaiani, C., Galet, L., Cuq, B., Desobry, S., Scher, J., (2011). Comparative
709
study of particle structure evolution during water sorption: Skim and whole milk powders.
710
Colloids and Surfaces B: Biointerfaces 87(1), 1-10.
711
Pisecky, J., (1997). Handbook of milk powder manufacture, Copenhagen.
712
Roetman, K., (1979). Crystalline lactose and the structure of spray-dried milk products as
713
observed by scanning electron microscopy. Netherlands Milk and Dairy Journal 33, 1-11.
714
Roos, Y., Karel, M., (1990). Differential scanning calorimetry study of phase transitions
715
affecting the quality of dehydrated materials. Biotechnology Progress 6(2), 159-163.
716
Roos, Y.H., Karel, M., (1991). Plasticizing Effect of Water on Thermal Behaviour and
717
Crystallization of Amorphous Food Models. J Food Sci 56(1), 775-777.
718
Schmidt, E.A., Law, D., Zhang, G.G.Z., (1999). Nucleation and Crystallization Kinetics of
719
Hydrated Amorphous Lactose above the Glass Transition Temperature. Journal of
720
Pharmaceutical Sciences 88(3), 291-296.
721
Sharples, A., (1966). Overall Kinetics of Crystallization. In: Sharples, A., editor. Introduction
722
to Polymer Crystallization, Edward Arnold Ltd, London, p 44–59.
723
Shimada, Y., Roos, Y., Karel, M., (1991). Oxidation of methyl linoleate encapsulated in
724
amorphous lactose-based food model. Journal of Agricultural and Food Chemistry 39(4),
725
637-641.
726
Soottitantawat,
727
Microencapsulation by spray-drying: Influence of emulsion size on the retention of volatile
728
compounds. J. Food Sci. 68(7), 2256-2262.
Ac ce p
te
d
M
an
us
cr
ip t
705
A.,
Yoshi,
H.,
Furata,
T.,
Ohkawara,
M.,
Linko, P.,
(2003).
Page 32 of 33
Maher Pg. 32 Soutari, N., Buanz, A.B.M., Gul, M.O., Tuleu, C., Gaisford, S., (2012). Quantifying
730
crystallisation rates of amorphous pharmaceuticals with dynamic mechanical analysis
731
(DMA). International Journal of Pharmaceutics 423, 335-340.
732
` {!!!$ ! $!['! -Lactoglobulin Interaction During
733
Storage of Model Whey Powders. Journal of Dairy Science 87(5), 1158-1166.
734
Tobin, M.C., (1974). Theory of phase transition kinetics with growth site impingement. I.
735
Homogenous nucleation. Journal of Polymer Science Part B: Polymer Physics 12, 399-406.
736
Urbanovivi, E., Segal, E., (1990). New formal relationships to describe kinetics of
737
crystasllisation. Thermochimica Acta 171, 87-94.
738
Yang, J., Grey, K., Doney, J., (2010). An improved kinetics approach to describe the physical
739
stability of amorphous solid dispersions. International Journal of Pharmaceutics 384(1–2),
740
24-31.
an
M te
744
Ac ce p
743
d
741 742
us
cr
ip t
729
Page 33 of 33