Journal of Food Engineering 114 (2013) 14–21

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Journal of Food Engineering

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Comparative study of deteriorative changes in the ageing of milk powder

Nima Yazdanpanah ⇑, Tim A.G. LangrishDrying and Process Technology Group, School of Chemical & Biomolecular Engineering, The University of Sydney, NSW 2006, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 May 2012Received in revised form 17 July 2012Accepted 27 July 2012Available online 4 August 2012

Keywords:Milk powderCrystallisationShelf-lifeProtein modificationEgg-shell particlesPhysical properties

0260-8774/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jfoodeng.2012.07.026

⇑ Corresponding author. Tel.: +61 02 93515661; faxE-mail address: nyaz3239@uni.sydney.edu.au (N.

Skim milk powders produced by spray drying (mostly amorphous) are commonly used in downstreamfood industries and have to be stored for long times after production before final use. However, theirphysicochemical qualities and properties decline during long-term storage due to the hygroscopicity ofamorphous lactose. Surface modification of particles by crystallisation of the outer layer, giving so called‘‘egg-shell’’ particles, has shown significant improvements in the physical properties of the powder intheir fresh state. In this study, the raw powder and processed powder (with modified surface) were storedat around 33% relative humidity and 25–30 �C for 30 weeks to investigate the effect of ageing on thesetwo types of powders. Agglomeration, large lactose crystal formation on the surface, surface compositionchanges and protein modifications were studied. The changes between the raw powder and process pow-der were compared after ageing. The non-hygroscopic crystalline surface layer showed significant bene-fits in maintaining the physicochemical qualities of the powders over long storage times. The aged rawpowder showed a 24% change in the crystallinity, a 3% change in the lactose/protein ratio on the surfaceand 6% protein denaturation compared with the aged processed powder with a 4% change in the crystal-linity, a 1.5% change in the lactose/protein ratio on the surface and 2% protein denaturation, respectively,after 30 weeks storage.

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1. Introduction 2005; López-Díez and Bone, 2000). Amorphous component of pow-

Milk powders are widely used in food processing industries orfor re-constituting and often must be stored for months. The shelflife for a food product is marked by an increase in undesirablephysicochemical qualities or microbial levels. Spray-dried milkpowders, in amorphous states, are very unstable. They have a ten-dency to sorb moisture and form caked powders that are not freeflowing. The increase in moisture content, due to the elevatedwater activity, causes bridge formation between particles, proteindenaturation and modification (Haque et al., 2010; Liu and Chau-dhary, 2011), the progress of Maillard reactions and decolouration(Bell, 2008), bacteria growth (Tapia et al., 2008) and quality loss(Sablani et al., 2007). These deteriorative changes start by sorbingmoisture form environment, leading to a decrease in the glass-transition temperature of amorphous components to below ambi-ent conditions, which initiates crystallisation and an increase inthe stickiness of the powders. The expelled water from crystallisa-tion further increases the water activity and enhances the changes.In addition, crystallisation of lactose from the matrix rearrangesthe protein–lactose bonds and de-stabilizes the proteins, meaningthat less amorphous lactose is available around the proteins to beattached to the H-bond active sites of the proteins (Buera et al.,

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: +61 02 93512854.Yazdanpanah).

ders readily sorb moisture from the environment, which leads tohigh moisture-content gradients near the surface, initiating mois-ture migration into the particles.

There are different methods that are commonly used to preventthe powders from sorbing moisture, such as keeping them in cooland dry conditions and using sealed impermeable packaging.Crystallisation of lactose in milk powders in pre-crystallisation orpost-crystallisation facilities has been suggested (Hynd, 1980;Yazdanpanah and Langrish, 2011b) to improve the powderstability against moisture sorption and enhancing the physicalproperties. Yazdanpanah and Langrish (2011b) showed theimprovement (decrease) in moisture sorption for the crystallisedlactose and milk powders that were crystallised in a fluidized-bed dryer/crystallizer. The mostly crystalline powders that wereproduced by that technique have a very much reduced tendencyto sorb moisture from the environment (even under very humidconditions for a long time).

Amorphous materials have greater solubility, porosity (Trivediand Axe, 2001) and bioavailability (Yang et al., 2010), comparedwith crystalline materials. The lower solubility and higher stabilityof the milk powders with crystallised lactose (mostly crystallineparticles) could be disadvantages when the powders are neededto be reconstituted or dispersed in dairy processing industries.Sandiness and rough texture in ice-cream and chocolate that ismade from this kind of crystallised milk powder are not desirablein sensory analysis (McSweeney and Fox, 2009).

N. Yazdanpanah, T.A.G. Langrish / Journal of Food Engineering 114 (2013) 14–21 15

The destabilisation effect of lactose crystallisation during stor-age and the elevated water activity also cause protein denaturation,releasing encapsulated ingredients, such as fat, and decreasing thenutritional value of the stored powder (Augustin et al., 2007; Fyfeet al., 2011). Proteins are stable in their low-energy, native (folded)state. Denaturation or unfolding, which are changes in the second-ary structures of proteins, increase the hydrophobicity of the pro-teins (Haque et al., 2010) and promote aggregation anddestabilisation (Allison et al., 1999). Most dairy powders haveshelf-lives of around 36 months in cool and dry conditions whenpacked in sealed moisture-proof containers. The moisture sorptionand diffusion rate, and consequential lactose crystallisation andprotein modification, increase the rate of quality loss in thepowders.

Most of the physical properties in spray-dried milk powders,such as flowablity, stickiness, agglomeration and caking, are sur-face-dominated properties (Fäldt and Bergenståhl, 1994;Yazdanpanah and Langrish, 2010). Surface amorphicity and com-position of the powder particles may then be expected to play animportant role in their flow behaviour, because flowability in-volves overcoming the surface attractions between the particles.Amorphous and hygroscopic materials on the surfaces have verysignificant effects on the agglomeration, stickiness and hygroscop-icity of the particles. Moisture sorption by crystalline lactose issignificantly lower than by amorphous lactose (Bronlund and Pat-erson, 2004), and this lower amount of moisture on the surfacecauses fewer moisture-induced changes inside particles. Also, par-ticles with crystalline surfaces have weak tendencies to formbridges with other particles due to the high thermodynamic stabil-ity of the crystalline state. Yazdanpanah and Langrish (2010) havedemonstrated that a thin layer of modified molecular structure onthe surfaces of particles can greatly improve the physical proper-ties of the powders. They created a new milk powder structure thatwas called an egg-shell structure, with a crystalline surface and anamorphous core, which has the good flowability and stability char-acteristics of crystalline powders, while preserving the desirablecharacteristics of the amorphous core. The previous report showssignificant improvements in stability, flowability and physicalproperties of the freshly-processed powders with egg-shell struc-tures compared with raw commercial powders, while maintainingthe same functionality. There are few reports in the literatureabout long-term studies of processed powders, and this study ad-dresses this issue.

The aim of this study was to investigate the water-inducedchanges in the skim milk powders during storage, comparing theraw commercial powder with the processed powders, with anegg-shell structure, during long storage. The effect of the crystal-line outer layer on the physical properties of the fresh powderhas been studied before (Yazdanpanah and Langrish, 2010); in thisresearch, the effects of the crystalline surface layer on the moisturesorption rate and the deteriorative changes in milk powder havebeen investigated.

2. Materials and methods

2.1. Materials

2.1.1. Raw skim milk powderSkim milk powder (medium heat) has been supplied by Murray

Goulburn Cooperative Co. Ltd. (Brunswick, VIC, Australia). Themanufacturer’s specification data sheet has showed that the com-position on a dry weight basis was lactose (59% w.w�1), protein(39% w.w�1) and fat (0.9% w.w�1). The moisture content of theraw milk powder has been measured to be 3.8% w.w�1 from ovendrying tests. This powder will be called raw powder hereafter.

2.1.2. Processed powderProcessed powder with egg-shell structure particles has been

made by a fluidized-bed crystallisation technique described before(Yazdanpanah and Langrish, 2010). The processing condition was60 �C, 40% RH and 20 min processing time. After processing, thepowder was transferred to a vacuum dryer and left under vacuumat room temperature for 4 h to slow down further crystallisation.The processed powder has been analysed by previously-describedmethods to confirm that it has an egg-shell type structure, and theamorphicity of the core of the particles has been checked.

2.2. Methods

2.2.1. Experimental setupThe raw powder and processed powder have been stored under

controlled humidity and temperature conditions at 25–30 �C and32–35% RH for 30 weeks. Both raw and processed powders havebeen kept separately in the same sorption box at these constanttemperature and humidity conditions for 30 weeks to age the pow-ders. The powders have been left under vacuum for 4 h to removethe initial moisture content prior to placing them in the sorptionbox containing saturated MgCl2 solutions giving �33% relativehumidity. A Tiny Tag Extra TGX3580 datalogger device from Gem-ini Data Loggers has been used for recording the temperature andthe relative humidity during the storage period. The equilibriummoisture content of skim milk powder at 33% relative humidityis 7.3% (w.w�1), and the glass-transition temperature was reportedas 22 �C (Schuck et al., 2005). The samples have been examinedwhen fresh and after 15 and 30 weeks of storage to assess anychanges in crystallinity and protein modifications. The tests after15 weeks storage time have showed that this time period wasnot adequate to develop the changes to an extent that was readilydetectable with the sensitivity of the equipment used here, such asXRD. Therefore the 30 weeks aged powders were compared withthe fresh state of the powders.

2.2.2. Scanning electron microscopyA scanning electron microscope was used to observe the surface

morphology of the powders. The samples were prepared by placinga small amount of each sample on a carbon tape that was placed onan aluminium sample disc. The sample was coated by a standard30 nm gold layer to produce the conductive surface (Emitech,K550X, Quorum Technologies, UK). The electron micrographs wereproduced using a Zeiss ULTRA plus (Carl Zeiss SMT AG, Germany)scanning electron microscope (SEM) in the In-Lens mode with anoperating voltage of 2 keV. A range of 500–30,000 times magnifica-tion was used in the images.

2.2.3. X-ray diffraction (XRD)X-ray diffraction (XRD) was used to investigate the bulk crystal-

linity of raw and processed powders by using a Siemens D5000 dif-fractometer. The scanning range was set to 5–30�, the step size was0.02� with a scanning rate of 1 step/s, and the operating conditionswere 40 kV and 30 mA. The EVA evaluation program (DIFFRACPlus, Bruker analytical X-ray system, GmbH) was used for peaksearching and calculating under-peak areas as part of the quantita-tive crystallinity analysis. Each scan has been repeated three times,and the average value and the standard deviation have beenreported.

2.2.4. Fourier transform infrared spectroscopy (FTIR)Attenuated total reflectance (ATR) spectra were acquired using

a single bounce diamond ATR (Universal ATR) in a nitrogen purgedNicolet 6700 FTIR spectrometer (Thermo Fisher Scientific Inc) con-trolled by OMNIC 8.2.387. The FTIR spectra were collected at a res-olution of 4 cm�1 with 32 scans over a range of wavelengths from

16 N. Yazdanpanah, T.A.G. Langrish / Journal of Food Engineering 114 (2013) 14–21

2000–500 cm�1. The collected data were analysed using the soft-ware Grams/AI 8.0 (Thermo Electron Corporation) for further dataprocessing, such as least-squares curve fitting and analysis (Gauss-ian + Lorenziane) and the calculation of the under-peak areas forquantifying the secondary structure of the proteins in the AmideI and Amide III regions. Second-derivative spectra were calculatedfrom normal spectra by means of the Savitsky–Golay algorithmwith a nine data point smoothing factor. The powders were keptin good optical contact by the diamond surface with a consistentapplication of low pressure. The powders have been kept undervacuum for four hours prior to scanning to remove the free mois-ture content and to minimize the effects of the H-bonds from watervapour. The analysis has been done for the shortest possible timeto avoid the particles being exposed to humid air. Each experimenthas been repeated three times, and the average value and the stan-dard deviation have been reported.

2.2.5. X-ray photoelectron spectroscopy (XPS)An XPS system of SPECS-XPS (SPECS, Germany) was used to

analyse the element concentrations and binding energies of theparticle surfaces. The XPS system was equipped with a PHOIBOS150-9 MCD energy analyser, and an Al K-alpha X-ray monochro-matic source (1486.74 eV) was used. Since we are using skim milkpowder, it is possible to assume the surface components aremainly proteins and lactose with small amounts of fat. Thereforethe equations from the literature (Fäldt and Bergenståhl, 1994)and the average of the standard component values for lactose, pro-tein and fat (Fäldt and Bergenståhl, 1994; Gaiani et al., 2006; Kimet al., 2005; Vignolles et al., 2009) were used to calculate the sur-face compositions of the powders.

2.2.6. Raman spectroscopyRaman spectroscopy was used to evaluate the crystallinity of

lactose on the surfaces of the particles. The Raman spectra werecollected using a Raman Station 400F (PerkinElmer, CA, USA). Thesamples were analysed using a power of 100% with a 785 nm laserand a five second exposure time, with five exposures over a rangeof wavelengths from 200 to 3200 cm�1. The spectra were analysedusing the software Spectrum v6.3.4.0164. The intensities of charac-teristic peaks of lactose at 355, 445, 865 and 1100 cm�1 were usedas references (Kirk et al., 2007; McGoverin et al., 2010; Murphyet al., 2005; Susi and Ard, 1974). Each experiment has been re-peated two times.

3. Results and discussion

3.1. Surface morphology and changes on the surface of the particles

The surface of the freshly processed powder has a crystallinetexture covered by fine lactose crystals, which were expected foregg-shell structure particles on the basis of previous work(Yazdanpanah and Langrish, 2010) (Fig. 1 FP). The particles of freshraw powder have smooth surfaces with no lactose crystals (Fig. 1FR). SEM micrographs show some morphological changes for thepowders during the storage time. Large lactose crystals have ap-peared on the surfaces of the raw powder particles. The particlesare agglomerated together substantially and the large lactose crys-tals have made bridges between particles. The amount and size ofthe lactose crystals formed during the storage time on the rawpowders (Fig. 1 AR) were larger than those on the aged processedpowders (Fig. 1 AP). The growth of lactose crystals on the surfacesof aged processed particles is evident, but the size and amount arevery small; there are no bridge formed between the particles andthe powders are still free-flowing.

The significant lactose crystal formation on the surface of theraw powder is due to the high availability of amorphous lactosethere, the hygroscopicity of amorphous lactose on the surfaceand the higher permeability of amorphous lactose. The hygroscopicamorphous lactose of the surface on the raw powder sorbs mois-ture and increases the water activity (Bronlund and Paterson,2004), make the molecules more mobile and lowers the glass-tran-sition temperature. This higher water activity and significantamount of expelled water from crystallisation on the surfacecauses other changes subsequently, such as component migrationor protein deformation, which will be discussed in detail in thecoming sections. The small lactose crystals on the surfaces of theaged processed powders might have been formed from joiningsmall lactose crystals that were formed in the processing stage.The fine lactose crystals might have acted as nuclei to increasethe crystal growth rate, but the low molecular mobility, becauseof the low amount of sorbed moisture and the high viscosity atthe higher glass-transition temperature, might have preventedthe formation of large crystals. The internal microstructure of theparticles appeared to remain similar during storage.

3.2. Crystallinity of the powders

The sorbed moisture on the surfaces of the particles and themoisture diffusion through the amorphous surface layers into theparticle core decreases the glass-transition temperature and vis-cosity and increases molecular mobility, which induces lactosecrystallisation. As SEM micrographs showed large lactose crystalson the surface of the aged powder particles (more on the surfacesof the aged raw powders), the surface crystallinity of the powdershave changed during the storage time. Surface crystallinity can bemeasured by Raman and FTIR spectroscopy; XRD is a technique fordetermining mainly the bulk crystallinity (Yazdanpanah andLangrish, 2010).

Kirk et al. (2007), Murphy et al. (2005), Seyer et al. (2000) andSusi and Ard (1974) found characteristic peaks for a- and b-lactosein their study, which are due to the characteristic stretching andbending vibrations of the COC bridge grouping. However, the char-acteristic and clear peaks are difficult to recognise in these spectra.The Raman spectra, shown in Figs. 2 and 3, show the changes incrystallinity of the powders after 30 weeks ageing compared withthe fresh powders. The surfaces of the fresh processed particles,which were covered by crystalline lactose, Fig. 2 (bottom curve),show sharp peaks at characteristic Raman shift points for lactoseat around 355, 445, 865, 1100 cm�1 (Murphy et al., 2005; Seyeret al., 2000).

The increase in the peak intensity with more surface crystallin-ity is expected, and while there may be a slight shift in the peak po-sition and changes in the sharpness/width of the peaks due todifferences in geometry, size and orientation of the newly-formed(or grown) crystals. It is very difficult to use Raman spectra in aquantitative way since there is no reference to which peak is moredominant for different amounts of crystallinity. Murphy et al.(2005) used the Raman depolarisation ratio to determine the sym-metry of a molecular vibration in the measured sample form andtried to numerically analyse the degree of crystallinity. Katainenet al. (2005) used the peaks at 440 and 470 cm�1 for evaluatingamorphous lactose in pure lactose (Katainen et al., 2005). The mea-surement of crystallinity based on their approach was not success-ful here, possibly due to the presence of other complex materials(proteins and minerals), which masked the crystals and sup-pressed/depressed the bond vibrations (intensity) causing a lackof repeatability for those regions. The same peaks at the sameplaces in the processed powders were observed (355, 445,865,1100 cm�1), but these peaks are weaker and have lower inten-sities, possibly due to smaller lactose crystals on the surfaces.

Fig. 1. (FR) Surfaces of fresh raw powder (fully amorphous). (AR) Aged raw powder after 30 weeks in storage. (FP) Surfaces of fresh processed powder, textured structure ofcrystalline outer layer with fine lactose crystals. (AP) Aged processed powder after 30 weeks in storage.

Fig. 2. Raman spectra of the processed milk powder in the fresh condition and afterageing, showing the similarity in the surface crystallinity. Fig. 3. Raman spectra of the raw milk powder in the fresh condition and after

ageing, showing the significant difference in the surface crystallinity.

N. Yazdanpanah, T.A.G. Langrish / Journal of Food Engineering 114 (2013) 14–21 17

Fig. 2 shows the processed powder for fresh and aged condi-tions. The spectra include the sharp peaks due to crystalline lactosein both samples with slightly higher intensities for the aged pro-cessed powders. In contrast, as Fig. 3 shows, the differences inintensity (and crystallinity) are much higher for the raw powderunder fresh and aged conditions. The difference is clearly signifi-cant (qualitatively) between the bottom line for the fresh raw pow-der and top line for the aged raw powder in the Fig. 3, when therepresentative peaks have higher intensity in the aged powderswith greater degree of crystallinity.

The surfaces of the raw powders after storage for 30 weeks havechanged significantly compared with the aged fresh powders. Sim-

ilar behaviours have been observed in the FTIR analysis of the pow-ders at 850–1200 cm�1 (results not shown here) where the agedraw powder has more noisy spectra with sharper peaks in the char-acteristic lactose region (Hogan and O’Callaghan, 2010). The sharppeaks at 1260, 900 and 875 cm�1 of the FTIR spectra have been re-ported to differentiate crystalline from amorphous lactose (Listioh-adi et al., 2009).

The XRD technique is a well-established method for quantifyingthe degree of bulk crystallinity and the crystal types in terms of thea/b lactose ratios in the powders. The most noted representativepeaks were located at 12.5�, 16.4�, 20.1� for a-lactose monohy-

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drate; 10.5�, 21� for anhydrous b-lactose; 18.2�, 19.1�, 21.1� for themixture of anhydrous a/b with a molar ratio of 5:3, and 19.5�,21.2�for mixture of anhydrous a/b with a molar ratio of 4:1 (Barhamet al., 2006; Gombas et al., 2002). The full spectra have not beenshown here, and relative crystallinity has been estimated accord-ing to the method of Nara and Komiya (1983), from the ratio ofthe peak areas, which is also called the ‘‘two-phase’’ method(Kim et al., 1997; LeCorre et al., 2011; Nara and Komiya, 1983),for different powders relative to the mostly-crystalline skim milkpowder as produced by techniques that have been described be-fore (Yazdanpanah and Langrish, 2011a). This summation doesnot take into account the recrystallisation between the differentpolymorphs. In this case, individual peaks should be considered.

Table 1 shows the measured bulk crystallinity for the fresh andaged powders by the XRD technique. The fresh raw powder wascompletely amorphous, and the spectrum was very noisy with nocharacteristic peak (not shown here), therefore the associated peakarea was assumed to be zero for the fresh raw amorphous powderin the X-ray spectra. The freshly processed powder has 8.9% crys-tallinity, which was mainly located on the surface of the particles,as Raman spectra shows and as described before (Yazdanpanahand Langrish, 2010). The aged raw powder showed 23.7 ± 0.1%crystallinity and the aged processed powder showed 12.7 ± 0.1%crystallinity. The crystallinity of the raw powder changed from0% to 23.7% and the crystallinity of the processed powder changedfrom 8.9% to 12.7%. It is difficult to determine the location of crys-tals and determine if all the crystallinity refers to the surface orsome crystals developed in the core of the particles. Since themoisture diffusion induces the crystallisation within the particlematrix, the crystal formation could have occurred inside the parti-cle. The change in crystallinity of the aged powders relative to theraw powder was 23.7 ± 0.1% compared with the 3.8 ± 0.1% changefor the aged processed powder. This change is significantly higherthan the processed powder and shows the effect of the non-hygro-scopic outer layer in decreasing moisture sorption and reducingthe diffusivity of moisture into the particle. T-tests have shownthat there are significant differences between the fresh and agedpowders at a 95% confidence level.

3.3. Surface composition

XPS (also known as ESCA) was used to determine the surfacecomposition of the powders. Milk powder is a multi-componentmixture, with lactose, protein and fat being the main components.The main purpose of using this technique was to measure thechanges in the lactose and protein concentrations on the surfaces.The powders were analysed before and after storage. The changesin the percentage compositions of different components on thesurface were calculated by a matrix formula method describedby Fäldt and Bergenståhl (1994) and Kim et al. (2009). Table 2shows the surface composition analysis for the different powdersin this study. In all the powders, the bulk fat was released to thesurface. The freshly-processed powder and aged powders havemore fat on their surface. This observation agrees well with arecent study for releasing fat during high-humidity and high-temperature processing for producing crystalline powders in a

Table 1Calculation of crystallinity by summation of peak areas under the characteristic peaks of

Powders Peak area Amount of crystallinity (%

Fresh raw 0 0Aged raw 32.1 ± 0.1 23.7 ± 0.1Fresh processed 12.0 ± 0.05 8.9 ± 0.05Aged processed 17.1 ± 0.1 12.7 ± 0.1Fully crystalline 135.0 ± 0.7 100

fluidized-bed dryer (Yazdanpanah and Langrish, 2012), increasingsurface fat content during storage of milk powder (Fäldt andBergenståhl, 1996) and releasing lipids in the storage of proteins(Gaiani et al., 2009). Fyfe et al. (2011) reported significant changesin the surface lactose content of milk protein concentrate (MPC85)after more than 90 days storage (Fyfe et al., 2011). The lactose con-centrations in the fresh raw powder and freshly processed powderwere similar, 37% and 36%, respectively, which shows restrictedmigration of the small molecules (lactose in this case) from thematrix to the surface; but the fat concentration is higher on theprocessed powder surfaces due to the melting of fats duringhigh-temperature processing in the fluidized-bed dryer. Thiscontrast could be due to the short processing time (30 min) whenlactose molecules have insufficient time to move to the surface,compared with the fat that diffuses in the liquid state with a higherdiffusion rate than solid lactose.

On this basis, the fresh raw powder and aged raw powders have37% lactose on the surface, but the lactose to protein ratios were74% and 77%, respectively. Statistical analysis show, that this threepercent change, which develops just 1.5 percent in the lactose con-centration, is significant compared with the aged processed pow-der. The processed powder was more stable during storage, interms of lower component migration. This stability again showsthe role of the non-hygroscopic crystalline layer in retaining theoriginal state of the matrix by reducing moisture diffusion. The re-sults have been supported by SEM micrographs that show fewerlactose crystals on the surfaces of the processed particles. Thiscomposition change could occur because of molecular migrationtowards the surface or inwards to the centre due to differencesin the solubilities of protein and lactose. The more soluble compo-nent (lactose in this case) could form a highly-saturated solutionby the sorption of moisture, diffuse to the surface and create morelactose crystals. Less soluble materials are likely to behave theother way (Meerdink and van’t Riet, 1995).

The increase in non-polar bonds, crystallinity and cross-linkedproteins on the surface can affect the rehydration rate of the parti-cles (Fyfe et al., 2011; Gaiani et al., 2006, 2009; Haque et al., 2010).The changes are associated with an increase in the hydrophobicityof the protein on the surface and the decrease in solubility of thelarge lactose crystals.

3.4. Protein conformation modification

Fourier transform infrared spectroscopy (FTIR) have been usedto study lactose crystallisation in milk powder (Lei et al., 2010;Listiohadi et al., 2009) and changes in protein secondary structures(Cai and Singh Bal, 1999; Chittur, 1998; Dong et al., 1990; Farrellet al., 2001; Goormaghtigh et al., 2009; Haque et al., 2010; Husonet al., 2011). Three broad peaks, with ranges around 1600–1700,1500–1600 and 1200–1320 cm�1 stand for the vibrations of amideI, II and III groups in proteins. The area between 800 and 1200 cm�1

presents the characteristic peaks of various C–O vibrations incarbohydrates.

Types of secondary structure of polypeptide chains, such as a-helices, b-sheets, b-turns, and random coils in protein, are most of-ten extracted from the IR spectra in the amide I region (Fig. 4),

XRD spectra for different powders.

of total) Change in crystallinity (aged-fresh/fully crystalline, %)

–23.7 ± 0.1–3.8 ± 0.1–

Table 2Different surface compositions of powders and atomic percentages of components detected by XPS analysis.

Powders Relative atomic concentration (%) Surface composition (%)

C O N Fat Protein Lactose L/Pa

Fresh raw 65.29 26.84 6.25 12 50 37 74.0Aged raw 66.56 26.83 6.00 15 48 37 77.0Fresh processed 66.73 26.54 6.12 15 49 36 73.4Aged processed 66.92 26.48 6.00 16 48 36 75.0

a Lactose to protein ratio.

Fig. 5. FTIR spectrum showing the amide III band of fresh-processed powder. Theouter envelope is the original spectrum, and the individual component peaksunderneath are the results of regression analysis. The peaks are associated withdifferent secondary structures.

N. Yazdanpanah, T.A.G. Langrish / Journal of Food Engineering 114 (2013) 14–21 19

which represents primarily the C@O stretching vibrations of amidegroups, and the amide III region, which has been described as aris-ing predominantly from C–N stretching vibrations coupled with N–H bending vibrations, with weak contributions from C–C stretchingand C@O bending (Bandekar, 1992; Cai and Singh Bal, 1999; Harisand Severcan, 1999). There is no H2O interference in the amide IIIregion; therefore, even though the signal from the amide III bandsare much weaker than from the amide I bands, the amide III regionis very useful for estimating protein secondary structure contents(Fig. 5). In this research, the populations of the secondary structureelements were studied in both the amide I and amide III regions.Fig. 6 shows the least-squares analysis for the consistency of thedata, which shows the secondary structure recognised from theamide I region supported by the results from the amide III region(R2 = 0.971).

Since a protein usually contains different secondary structuralelements, such as a-helices, b-sheets, b-turns, and random coils,the amide I and III bands are composite bands consisting of over-lapping signals. Fourier self-deconvolution or second derivativescan be used to identify the number and the positions of the bandsunderlying these amide bands. Generally, the second derivative IRspectra can enhance the resolution and amplify even small differ-ences in ordinary IR spectra. Least-squares curve fitting techniquesare particularly useful for precisely following changes in proteinconformations as a function of adsorption time. The techniquehas been used for quantitative estimation of different secondarystructures in proteins as a linear combination of individual compo-nent bands, by iterative adjustment of the heights, widths, andpositions of these bands. The fractional areas of the individualbands give the fraction of the secondary structure elements. Thisprocedure has provided a very good estimate of the protein sec-ondary structure (Chittur, 1998; Haris and Severcan, 1999). There-fore the percentages of b-sheets were calculated by adding the

Fig. 4. FTIR spectrum showing amide I bands of fresh processed powder. The outerenvelope is the original spectrum, and the individual component peaks underneathare the results of regression analysis. The peaks are associated with differentsecondary structures.

Fig. 6. PLS regression of amide I vs amide III percentage for the population ofdifferent structural components in the secondary structure of proteins.

areas of all bands assigned to the b-sheets and expressing the ratioof this sum as a fraction of the total band area, as the population ofb-sheets. The same procedure has been carried on for b-turns anda-helices in the amide I and III regions. The peak assignment ofdeconvolved amide I bands (1700–1600 cm�1) and amide III bands(1320–1200 cm�1) was done using the procedures outlined in pre-vious reports (Bandekar, 1992; Cai and Singh Bal, 1999; Chittur,1998; Dong et al., 1990; Farrell et al., 2001; Haris and Severcan,1999; Singh Bal, 1999). The results for the fresh processed powderhave been shown in the Figs. 4 and 5, where Fig. 4 shows the amideI bands of the freshly processed powder and the associated bandsfor the different secondary structural elements; and Fig. 5 showsthe amide III bands of freshly-processed powder with the assigned

Table 3Population (relative percentages) of different structural components in the secondary structure of the proteins in amide I and amide III regions for different powders.

Powders Amide I Amide III

a-Helices b-Sheets b-Turns a-Helices b-Sheets b-Turns

Fresh raw 28.0 ± 0.05 52.9 ± 0.1 19.1 ± 0.1 26.1 ± 0.05 53.8 ± 0.1 20.5 ± 0.05Aged raw 22.1 ± 0.1 57.0 ± 0.05 21.0 ± 0.2 20 ± 0.1 56.2 ± 0.1 24.1 ± 0.2Differences in raw powder �6 �4 �1.9 �6 �2.5 �3.6Fresh processed 26.3 ± 0.05 53.8 ± 0.1 20.1 ± 0.1 24.8 ± 0.1 54.5 ± 0.3 20.9 ± 0.1Aged processed 24.0 ± 0.1 54.7 ± 0.1 21.4 ± 0.1 22.4 ± 0.3 55.3 ± 0.1 22.5� ± 0.3Differences in processed powder �2.3 �0.9 �1.3 �2.4 �0.8 �1.6

20 N. Yazdanpanah, T.A.G. Langrish / Journal of Food Engineering 114 (2013) 14–21

bands. The peak at�1648 cm�1 was considered to represent a loopor helix and the peak at �1658 cm�1, a large loop and assigned asan a-helix and the peak of �1650 cm�1 as an a-helix in the fittingroutine for the amide I region; and in the amide III region, the peakat �1305 cm�1 was assigned as an a-helix. The full list of the peakpositions for all the secondary structures of polypeptide chains,such as a-helices, b-sheets, b-turns, and random coils in protein,are available in the before-mentioned references.

The FTIR analyses of the amide I and III regions show somechanges in the secondary structure of the proteins, as shown inTable 3. The transformations (unfolding) of helical and loop com-ponents (folded structure) to sheet and turn components indicatethe changes in the secondary structure of the milk proteins inthe powders after storage. The decrease in the helical componentsand a corresponding increase in the b-components is significant interms of the ageing of the powders due to the protein conforma-tional modifications and the destruction of the secondary structure(native structure) of the proteins. After long storage, the proteinsare not completely denatured since they are in the solid form,but storage in high water activity conditions caused some changes,in terms of denaturation, modification or cross-linking. The pres-ence of the shoulders, the broadened bands, the appearance of fur-ther bands and misplaced bands may be attributed to theseaggregated structures, but the exact explanations are unclear.The abnormal bands were mostly detected in the aged powders.Mauerer and Lee (2006) reported some unusual peaks associatedwith intermolecular H-bonding in extended polypeptide chains,non-H-bonded amide groups and high-frequency inter- or intra-molecular H-bonding for spray-dried Poly-L-lysine powder (Mau-erer and Lee, 2006). T-tests have shown that there are significantdifferences between the fresh and aged powders at a 95% confi-dence level for each amide I and amide III regions separately. Inthe raw powder, the population of a-helices significantly de-creased in the aged raw powder (�6%) compared with the freshraw powder. The amide I shows the decrease from 28.0 ± 0.05%in the fresh raw powder to 22.1 ± 0.1% in the aged raw powder,and amide III shows the same 6% decrease in a-helices but from26.1 ± 0.05% to 20 ± 0.1% in the aged powder. The population ofb-sheets and b-turns increased significantly in the aged powder(Table 3). The amide I region showed a 4 % increase in b-sheetsand a 1.9% increase in b-turns , while the amide III region showed2.5% increase in b-sheets and a 3.6% increase for b-turns. In theaged processed powder, the decrease in the amount of a-heliceswas around 2.3%,the amount of b-sheets increased by about 0.9%and the b-turns increased by the average of 1.5% (the amide Ishowed a 1.3% increase and the amide III region showed a 1.6%).The reason for the differences in the amount of b-sheets andb-turns between the amide I and amide III regions was not clear,but it could be due to multiple bands existing in these regions.The results for a-helix have been very repeatable for all the pow-ders in the amide I and III, which were used as reference pointsfor assessing the changes in the secondary structure of the proteins(unfolding). The reduction in the a-helices content of the pro-cessed powder has been significantly lower than the populationof the a-helices in the raw powder, which suggests more loss of

the native structure for the proteins in the raw powder during stor-age compare with the processed powders. There was a slightchange in the secondary structure of the protein during processingat high temperatures and humidities, which was represented bythe decrease in the a-helix population from the raw to the pro-cessed powder from 28% to 26.3%. However, the results of the agedpowders showed that the processed powder maintains the nativestructure of the proteins better, since the relative fraction ofa-helices in the aged processed powder is higher than in the agedraw powder, and consequently the relative fractions of b-sheetsand b-turns are lower. The a-structure to b-structure ratio hasbeen used as a reference for the native structure of the proteins,and a decrease in helical components has been reported as indicat-ing the loss of the native secondary structure (Chittur, 1998; Goor-maghtigh et al., 2009; Haris and Severcan, 1999; Huson et al.,2011). The role of the crystalline surface layer in maintaining thesecondary structure of the proteins by keeping low internal wateractivity appears to have been significant during storage.

4. Conclusions

This research has assessed the effect of storage on raw skimmilk powders and processed powders, particularly regarding thecharacterisation of the powder surface. The paper reports the roleof the crystalline surface layer of the processed powder in prevent-ing (or slowing) deteriorative changes in the particles. The hygro-scopicity of amorphous lactose on the surface of the raw powderand the higher permeability of amorphous lactose cause moisturesorption and increase the water activity, which provides highermobility for the molecules and lowers the glass-transition temper-ature. This higher water activity and large amount of expelledwater from crystallisation on the surface cause other changes sub-sequently, such as component migration and/or protein deforma-tion. The processed powder with a higher amount of crystallinelactose on the surface, has been found to sorb less moisture reduc-ing these subsequent material changes. Surface analysis reports,crystallinity, composition and protein modification have all indi-cated that there were much smaller changes in the ageing of theprocessed powder in contrast to significant changes in the agedraw powder.

Acknowledgements

The authors would like to thank Dr. Elizabeth Carter from theSchool of Chemistry, University of Sydney for her helps with theFTIR analysis. The authors would also like to thank Murray Goul-burn Cooperative Co. Ltd. for providing the dairy powders for thisresearch.

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