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Keywords:Milk fat globules

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ecent studies showedhe outer bilayer of theingomyelin in domains2011; Zou et al., 2012).ols (TAG; esters of fatty

Food Research International 54 (2013) 15411552

Contents lists available at ScienceDirect

Food Research

e lsof the total energy intake (German & Dillard, 2006; Jensen et al.,1995,; Jensen, 1999). Lipids are secreted in breast milk in the form ofunique delivery systems called milk fat globules (Hamosh et al., 1999;

acids and glycerol) that account for at least 98% of milk lipids (German &Dillard, 2006; Jensen et al., 1995; Jensen, 1999). Mature human milkcontains 11 major fatty acids, mostly long-chain fatty acids (C16:0;of human milk for infants, the role played by the structure of itscomponents and their physical state, particularly those of lipids, arefar from being fully elucidated and need to be further investigated.

Mature human milk contains 3 to 5% lipids that represent the mainsource of energy for the breast-fed infant, contributing some 40 to 55%

(German & Dillard, 2006; Jensen et al., 1995). Rthe heterogeneous distribution of polar lipids in thumanMFGM, with the lateral segregation of sphthat could have functional roles (Lopez &Mnard,The core ofmilk fat globules contains triacylglycerthe nutritional requirements of the infant (nutrients in an optimalratio, i.e. proteins, carbohydrates and lipids) and health benets. Breastmilk is also a tool by which the mother continues her protection fromthe fetus to the newborn child, by supplying the necessary instructionsfor the development of the intestinal mucosa and herewith also for thedevelopment of the immune and nervous system, as well as for thecorrect activity of the metabolic machinery. Although the importance

size distribution of human mature milk fat globules ranges from 1 to10 m with an average diameter between 4 and 5 m (Lopez &Mnard, 2011; Regg & Blanc, 1981; Zou et al., 2012). Milk fat globulesare surrounded by a biological membrane, called the milk fat globulemembrane (MFGM), which is rich in phospholipids, sphingolipids(mainly sphingomyelin), glycosphingolipids (cerebrosides, gangliosides),glycoproteins (mucins, lactadherin), proteins, cholesterol, and enzymesreview Lopez, 2011). Milk fat globules correspsupramolecular assemblies which are secrete

Corresponding author at: INRA, UMR STLO, 65 rue deFrance. Tel.: +33 2 23 48 56 17; fax: +33 2 23 48 53 50.

E-mail address: Christelle.Lopez@rennes.inra.fr (C. Lop

0963-9969/$ see front matter 2013 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.foodres.2013.10.014t breast feeding provides

of the mother to vehicle lipids and other bioactive molecules in thegastrointestinal tract of the newborn (i.e. fat-soluble vitamins,phospholipids, sphingolipids, cholesterol, membrane proteins). TheIt has been recognized for a long time thaCrystallization in emulsionSolid fat phaseMelting proleX-ray diffractionDifferential scanning calorimetry

1. Introductionand health, their physical properties remain poorly known. In this study, the crystallization and meltingproperties of breast milk triacylglycerols (TAG; 98% of milk lipids) were investigated in fat globules and inanhydrous state. The thermal and structural properties of breast milk TAG were characterized by differentialscanning calorimetry and synchrotron radiation X-ray diffraction, respectively. Breast milk fat globules have amean diameter of 4 to 5 m. They contain 4857% saturated fatty acids with 2729% of C16:0. Storage of breastmilk at 4 C in the refrigerator leads to the crystallization of high-melting point TAG within fat globules in 2L(41.7 ) lamellar structures. The nal melting point of these TAG crystals, Toffset = 41.1 1.6 C, is above in-body temperature of infants. Hence, the solid TAG phase could affect the mechanisms of breast milk fat globulesdigestion and absorption for T b Toffset of TAG crystals, by decreasing the rate of TAG hydrolysis andsolubilization.

2013 Elsevier Ltd. All rights reserved.Accepted 7 October 2013energy and bioactive molecules to infants. Although they are important in newborn infant nutrition, growthArticle history:Received 19 August 2013

Breast milk fat globules areSolid triacylglycerols within human fat glomelting point above in-body temperaturestorage of breast milk at low temperature

Christelle Lopez a,b,, Valrie Briard-Bion a,b, Claudie Boa INRA, UMR 1253 STLO, 65 rue de Saint-Brieuc, F-35042 Rennes cedex, Franceb Agrocampus Ouest, UMR 1253 STLO, 65 rue de Saint-Brieuc, F-35042 Rennes cedex, Francec CNRS, UMR8612 Universit Paris-Sud XI, 5 rue J.-B. Clment, F-92296 Chtenay-Malabry, Frad Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin BP48, F-91192 Gif-sur-Yvette, France

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j ourna l homepage: www.ond to the well-denedd by the epithelial cells

Saint-Brieuc, F-35042 Rennes,

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natural colloidal assemblies secreted by lactating mothers to provide dietary

International

ev ie r .com/ locate / foodres90% of fatty acids) derived from the circulation and medium-chain fattyacids (C8:0 to C14:0) synthetized de novo within the mammarygland (Bitman, Wood, Hamosh, Hamosh, & Mehta, 1983; Jensen,1999). Human milk lipids also supply essential polyunsaturatedfatty acids (PUFA), including linoleic acid of the n6 series(LA, 18:2 n6) and -linolenic acid of the n3 series (ALA, 18:3

the sn-2 position (Jensen, 1999; Zou et al., 2013a). This specic internalstructure of human milk TAG is of particular importance for their

1542 C. Lopez et al. / Food Research International 54 (2013) 15411552hydrolysis by lipases and their absorption (Lindquist & Hernell, 2010).The fatty acid composition of fats, i.e. the amount in long-chain

saturated fatty acids, and the internal structure of TAG are importantparameters governing their physical properties, particularly theircrystallization properties. Information about the thermal properties ofanhydrous human milk TAG is scarce (Zou et al., 2013b). Investigationof the crystallization of TAG in emulsion droplets is much morechallenging than in anhydrous state. Research studies focused on thecrystallization properties of milk TAG dispersed within fat globulesfrom various species, e.g. bovine milk (Lopez, Bourgaux, Lesieur, &Ollivon, 2007; Sderberg et al., 1989; Walstra, 1967), goat milk(Ben Amara-Dali, Lopez, Lesieur, & Ollivon, 2008), dromedary milk(Lopez, Karray, Lesieur, & Ollivon, 2005). However, little attentionhas been paid to the physical properties of TAG within human milkfat globules.

Breast milk is produced in the mammary gland and directlytransferred during nursing from the mother to the infant, at the in-body temperature. Mothers are also able to express their milk bypumping and fed their infant with a bottle. Pumping and storing breastmilk allows having a supply of milk for the newborn when it is needed.Collecting breast milk during the day while at the ofce can be a goodoption for moms who return to work outside the home after having ababy. It is also useful for mothers who need to bring breast milk totheir infant staying at the hospital. In the case when breast milk isnot consumed just after collection, storage of expressed breast milkneeds to be performed at room temperature (1926 C) for up to 8 h(Hamosh, Ellis, Pollock, Henderson, & Hamosh, 1996), in a refrigerator(4 C) for up to 5days (Sosa & Barness, 1987) and in a freezer (18 C)up to 6 months (Berkow, Freed, Hamosh, et al., 1984; Friend, Shahani,Long, & Vaughn, 1983). Storage of breast milk at low temperatureprevents chemical deterioration (e.g. lipid peroxidation of PUFA,proteolysis, lipolysis) and microbiological risks (e.g. development ofpathogenic bacteria). Also, storage of breast milk at low temperature(i.e. for T b Tbody) raises the question of the crystallization propertiesof human milk TAG. Indeed, previous studied reported the formationof a solid TAG phase upon storage at 4 C of bovine milk fat globulesand emulsion droplets (Bugeat et al., 2011; Lopez, Bourgaux, Lesieur,& Ollivon, 2002). Therefore, there is a need to gain knowledge abouthuman milk fat globule properties as a function of temperature.

The objectives of this studywere to investigate the thermal propertiesof mature human milk fat and to describe the crystalline structuresformed by human TAG upon storage of breast milk at 4 C. The thermaland structural properties of human TAG were characterized usingdifferential scanning calorimetry and high-ux radiation synchrotronX-ray diffraction, respectively.

2. Materials and methods

2.1. Breast milk samples and anhydrous fat

Mature human milks have been collected from volunteer healthywomen with 2 to 7.5 months post-partum, living in the west part ofFrance (French Britany). Following their secretion from the lactatingcells, expressed milks were removed from the mammary gland with an3) and the long-chain PUFA (ARA, n6;DHA, n3;DPA, n3 andEPA, n3), that have been related to functional outcomes of therecipient infants such as visual acuity and development of cognitivefunctions during the rst year of life (Koletzko et al., 2001). Fatty acidsare distributed in the 3 positions of the glycerol backbone, leading to alarge amount of TAG molecular species that have been characterizedin colostrum, transitional milk and mature milk (Zou et al., 2013a). Inhuman TAG, greater than 60% of the palmitic acid C16:0 is located inbreast milk pump, as already reported in Lopez and Mnard (2011).Anhydrous fats were obtained from breast milks by an adaptedprotocol of the cold extraction procedure developed by Folch, Lees,and Stanley (1957) (see Section 2.4.).

2.2. Fat globule size measurements

The size distributions of human milk fat globules were determinedby laser light scattering using aMastersizer 2000 (Malvern Instruments,UK) with two laser sources. The refractive indexes used were 1.458 and1.460 for milk fat at 633 nm and 466 nm respectively and 1.333 forwater. The milks have been heated to 371C before the experiments.About 0.2 mL of mature milk was diluted in 100mL of water directlyin the measurement cell of the apparatus in order to reach 10%obscuration. The casein micelles were dissociated by adding 1 mL of35mM EDTA/NaOH, pH7 buffer to themilks, in the apparatus. Standardparameters were calculated by the software, as described in Lopez andMnard (2011). The experiments were performed in triplicate for eachbreast milk.

2.3. Confocal laser scanning microscopy

The microstructural analysis of the milks was performed with aninverted microscope NIKON Eclipse-TE2000-C1si (NIKON, Champignysur Marne, France) allowing confocal laser scanning microscopy (CLSM)and optical microscopy using differential interferential contrast (DIC,also called Nomarski contrast). Confocal experiments were performedusing aHeNe laser operating at 543nmwavelength excitation (emissionwas detected between 565 and 615 nm). The observations wereperformed using 40 (NA 1.33) and 100 (NA 1.4) oil immersionobjectives. Triacylglycerols were stained with the lipid-soluble Nile Reduorescent dye (5H-benzo phenoxazine-5-one, 9-diethylamino;Sigma-Aldrich, St Louis, USA). After addition of the uorescent probeinto the milks, the samples were kept at 4 C up to 48 h before themicrostructural analysis. Then, 5 L of the samples stained with theuorescent probe were deposed onto the glass and observed on themicroscope. The microstructural analyses were performed at 4 1 C,using a temperature-regulated stage (Linkam Scientic Instruments Ltd.,Tadworth, Surrey, England). DIC images superimposed to the emissionuorescent recorded in the CLSM images. The two-dimensional imageshad a resolution of 512 512 pixels and the pixel scale values wereconverted into micrometers using a scaling factor.

2.4. Fatty acid composition

An adapted protocol of the cold extraction procedure developed byFolch et al. (1957) was used for the extraction of total lipids from themilks. Two grams of breast milk samples were homogenized withchloroform/methanol (2:1, vol/vol) with a mixer (IKA, Fisher Bioblock).The extractwas shaken and equilibratedwith one-fourth its volume of asaline solution (NaCl=0.73%, w/w). The upper phasewaswashed threetimes with a mixture of 40 mL of chloroform/methanol (2:1, vol/vol)and 10 mL of 0.58% (w/w) NaCl. The solvent phases containing totallipids were ltered (Whatman lter paper, 2.5 m, Grosseron, France)through a pinch of anhydrous sodium sulfate and evaporated undervacuum. The extraction of total lipids was performed in duplicate ortriplicate to obtain a coefcient of variation b5%. Total lipids extractedwere stored at20C until further gas chromatography (GC) analysis.Overall fatty acid compositions of human milks were determinedaccording to the method adapted from Park and Goins (1994). Thisconsisted in the conversion of initial TAG into fatty acid (FA) methylesters which were then analyzed by GC. This was performed by usingan Agilent 7890 GC system (Agilent Technologies France, Massy, France)with instrument parameters already reported by Lopez et al. (2008), i.e.with 2 capillary columns (50 m by 0.32 mm; lm thickness = 0.25 meach one) coated with 70% cyanopropyl polysilphenylenesiloxane (BPX-

70, SGE, Ringwood, Vic., Australia) mounted in series. Experimental

1543C. Lopez et al. / Food Research International 54 (2013) 15411552conditions were as follows: initial temperature of on-column injection(1 L) was 40 C for 0.2min; the temperature injector was programmedto increase to 200 C at a rate of 200 C/min, an isotherm at 200 C for6 min and a decrease in temperature to 40 C at a rate of 200 C/min.The Flame Ionization Detector temperature was 250 C; carrier gas washydrogen at a pressure of 138 kPa. Oven temperature was programmedas follows: 50C for 10min followed by an increase to 175 C at a rate of10 C/min; the oven was maintained at this temperature for 27 min.Then, temperature was increased to 215 C at a rate of 4 C/min andmaintained during 16min. Total analysis time was 75.50min. Retentiontimes were determined by injection of commercial mixes of FA methylester standards (Larodan Fine Chemicals, Malm, Sweden) and trans FAwhich are all purchased from Sigma-Aldrich (Saint Quentin Fallavier,France). The analyseswere performed in triplicate from two independentsamples.

2.5. Differential scanning calorimetry (DSC) experiments

DSC measurements were performed with a DSC Q1000 (TAInstruments, Newcastle, DE). Apparatus calibration was made withindium standard (156.66 C and 28.41 J/g melting temperature andenthalpy variation, respectively). Total sample masses, typically rangingfrom 9 to 16 mg, were introduced in 20 L aluminum pans whichwere hermetically sealed after sample introduction. An empty andhermetically sealed pan was used as reference.

To characterize the thermal properties of anhydrous breast milk fat,the samples were i) melted at 60 C and introduced in aluminum pans,ii) stored at 60 C for 5min, iii) cooled down to 60 C at 2 C/min inorder to achieve complete crystallization of the TAG and iv) heated to60 C at 2 C/min. The experiments have been performed in triplicatefor each sample.

To characterize the thermal properties of TAG dispersed in breastmilk fat globules, two different protocols were used. The rst protocolaimed at characterizing the thermal properties of TAG after eliminationof their thermal history (i.e. melting of TAG before a cooling kinetic).The samples were i) introduced in the DSC apparatus and heated at60 C, ii) stored at 60 C for 5min, iii) cooled down to5 C at 2 C/minand iv) heated to 60 C at 2 C/min. The second protocol aimed atcharacterizing the thermal properties of TAG after storage at 4 C. Thesamples were i) stored at 4 C for 48h, ii) introduced in the calorimeterpre-cooled to 4 C, iii) equilibrated at 4 C for 30 min, and iv) cooledfrom 4C to5C at 2C/min to avoid the interference of the launchingof the calorimeter prior to heating from5 C to 60 C at 2 C/min. Theexperiments have been performed at least 3 times for each sample,using independent pans in order to avoid the physical destabilization ofbreast milk fat globules.

Thermal parameters (i.e. Tonset, Toffset) were determined by usingUniversal Analysis 2000 v 4.1 D software (TA Instruments, Newcastle,DE).

2.6. Time-resolved and temperature-controlled synchrotron X-ray diffraction(XRD) experiments and XRD/DSC coupling

X-ray diffraction (XRD) experiments coupled to high-sensitivitydifferential scanning calorimetry (DSC) were performed at SOLEILsynchrotron (Gif-sur-Yvette, France) on the SWING beam line. Theset-up of the XRD/DSC coupling is presented in Fig. 1. XRD data werecollected by a two-dimension detector. The diffracted intensity wasreported as a function of the scattering vector q= 4sin/ where 2is the scattering angle and is the wavelength of the incident beam( = 0.828 ; 15 keV). The sample-to-detector distance was setto 554 mm to allow recording of XRD patterns in the q-range from0.08 1 to 1.8 1, thus covering the small- and wide-angle regionsof interest for TAG molecules. Calibration of the q-range was carriedout with pure -tristearin (Lavigne, Bourgaux, & Ollivon, 1993) for

wide angles and silver behenate (Blanton, Barnes, & Lelental, 2000) forsmall angles. Intensity values were normalized for beam intensity,acquisition time and sample transmission. Each XRD pattern recordedas a function of time or temperature displayed concentric signal ringswhich were integrated circularly to yield the intensity versus q.

The breast milk samples (around 20 L) were introduced intothin quartz capillaries of 1.5 mm diameter (GLAS W. Muller, Berlin,Germany) and stored for 48 h at 4 C. Then, the capillaries were in-troduced within a specially designed calorimeter (Microcalix, Ollivonet al., 2006) pre-cooled to 4 C, which was inserted in the beampathway. The samples were characterized in isothermal conditions at4 C; 30 XRD patterns were acquired at 4 C, for 6 s each (4.5 s lifetime; 1.5 s dead time) and summed to obtain an average XRD patternof 180s (135s life time; 45 s dead time). Then, the breast milk sampleswere submitted to controlled heating at a rate of 2 C/min from 4C upto 60 C while differential scanning calorimetry (DSC) signals and XRDpatterns were simultaneously recorded as a function of time. Duringtemperature scan, XRD patterns (186 total number) were recordedevery 6 s (4.5 s life time; 1.5 s dead time). For graphic representation,each XRD pattern corresponded to the sum of successive ones. DSCdata were collected using a home-made computing system. Temper-ature calibration of the calorimeter was ensured by using pure lauricacid as standard (43.7 C melting temperature, Grabielle-Madelmont &Perron, 1983). The precision of temperature regulation was 0.1 C.The samples have also been characterized after storage at roomtemperature: 30 XRD patterns were acquired at 19 C in isothermalconditions, for 6 s each (4.5 s life time; 1.5 s dead time) and summedto obtain an average XRD pattern of 180 s (135 s life time; 45 s deadtime).

Structural parameters were determined from the XRD patterns usingPeakFit software (Jandel Scientic, Germany). Position, integratedintensity (area) and maximum intensity of the Bragg reections werededuced from t with Gaussian and Lorentzian sum (amplitude)function (Bugeat et al., 2011).

3. Results and discussion

3.1. Fatty acid composition of mature breast milks

Themature breast milks characterized in this study contained 48.4 to56.7wt.% saturated fatty acids (Table 1). The main saturated fatty acidswere C16:0 (26.629.7%), C18:0 (7.410.5%) and C14:0 (6.210.2%).The main monounsaturated fatty acid was C18:1 c9 (30.136.2%). Themain PUFA were the essential fatty acids C18:2 n6 (LA; 6.39.0%)and C18:3 n3 (ALA; 0.480.97%) and the long-chain PUFA ARA(C20:4 n6; 0.220.49%), DPA (C22:5 n3; 0.100.17%) and DHA(C22:6 n3; 0.110.33%). Our results are in agreement with literature(Jensen, 1999; Smit, Martini, Mulder, & Boersma, 2002). The fatty acidcomposition of breast milk TAG varies with such factors as dietary habitsof lactating women, gestational age, lactation stage and genetics (Jensen,1999). The major proportion of milk lipids is formed from circulatinglipids derived from the maternal diet, and from maternal body stores.Part of the milk lipids can be synthetized de novo in the mammarygland fromglucose, which results primarily in the formation of saturatedfatty acidswith 10 to 14 atoms of carbon (Thompson& Smith, 1985). Theessential fatty acids LA and ALA must at some time be derived from thematernal diet since they cannot be synthetized de novo in the humanorganism.

3.2. Thermal properties of anhydrous human milk fat

The thermal properties of the fat extracted from breast milksexpressed by 5 different mothers were investigated in the range 60 to60 C on cooling at 2 C/min and then on subsequent heating at2 C/min (Fig. 2). Similar DSC proles have been recorded for thedifferent breast milk fats. On cooling, the DSC thermograms show the

successive formation of at least two exotherms that correspond to the

Fig. 1. Experimental set-up allowing the coupling of high-sensitivity calorimetry (microcalorimeterMicrocalix) with time-resolved synchrotron X-ray diffraction in the Swing beamline atSOLEIL synchrotron (Gif-sur Yvette, France). The calorimeter is positionedwith the capillary containing the sample perpendicular to the beam in suchaway that thediffraction patterns arerecorded in the vertical plane by one two-dimension detector at small and wide angles. Each X-ray diffraction pattern recorded as a function of time or temperature displayed concentricsignal rings which were integrated circularly to yield the intensity versus scattering vector q. Nanovoltmeter (nVmeter) and temperature controller (T control) are monitored by a singlecomputer. The temperature-controlled cryostat (TCC) is kept at constant temperature (e.g. 4 C).

Table 1Fatty acid composition of mature human milks.

Fatty acids (wt.%) a Mature human milks

#1 #2 #3 #4 #5 range

C8:0 0.16 0.01 0.160.01 0.170.01 0.150.01 0.170.01 0.140.18C10:0 1.17 0.03 1.200.02 1.210.03 1.100.03 1.200.06 1.071.26C12:0 5.56 0.06 6.410.04 4.310.03 3.990.08 4.110.12 3.996.45C14:0 9.32 0.07 10.120.10 7.820.05 7.700.10 6.260.08 6.1810.22C14:1 c9 0.44 0.01 0.410.01 0.500.01 0.540.01 0.380.01 0.370.55C15:0 0.58 0.01 0.520.01 0.640.01 0.630.01 0.490.01 0.480.65C15:1 0.14 0.01 0.120.01 0.160.01 0.160.01 0.110.01 0.100.17C16:0 26.84 0.23 26.550.19 28.640.17 29.500.22 27.500.03 26.629.7C16:1 1.70 0.01 1.520.02 2.300.03 2.500.03 2.960.04 1.503.0C17:0 0.44 0.01 0.420.01 0.560.01 0.480.01 0.400.01 0.390.57C17:1 0.23 0.01 0.220.01 0.330.01 0.270.01 0.270.01 0.210.28C18:0 9.26 0.16 10.330.16 9.990.10 9.560.21 7.570.16 7.410.5C18:1 t6-7-8-9 0.23 0.15 0.310.15 0.200.08 0.170.04 0.160.08 0.080.46C18:1 t10+11 0.85 0.22 0.920.17 1.230.20 0.680.15 0.400.02 0.381.43C18:1 c9 32.21 0.16 30.270.14 32.670.25 32.010.10 36.010.18 30.136.2C18:2 t9t12 0.20 0.04 0.180.06 0.280.06 0.140.05 0.120.02 0.100.34C18:2 n6 (LA) 7.45 0.06 7.030.07 6.390.08 7.470.03 8.960.04 6.39.0C18:3 n3 (ALA) 0.83 0.01 0.960.01 0.530.01 0.700.01 0.490.01 0.480.97C20:0 0.47 0.04 0.480.01 0.610.04 0.500.01 0.350.02 0.330.65C20:4 n6 (ARA) 0.28 0.01 0.270.01 0.230.01 0.310.01 0.480.01 0.220.49C22:5 n3 (DPA) 0.16 0.01 0.130.01 0.080.01 0.110.01 0.130.01 0.100.17C22:6 n3 (DHA) 0.32 0.01 0.260.01 0.110.00 0.190.01 0.220.02 0.110.33SFA 54.2 0.2 56.7 0.3 54.4 0.1 54.0 0.3 48.4 0.1 48.357.0LC-SFA (C16:0C24:0) 37.4 0.1 38.3 0.2 40.2 0.1 40.4 0.1 36.1 0.2 35.940.5UFA 45.8 0.2 43.3 0.3 45.6 0.1 46.0 0.3 51.6 0.1 43.051.7MUFA 35.9 0.1 33.9 0.3 37.4 0.1 36.4 0.2 40.4 0.1 33.640.5PUFA 9.8 0.1 9.4 0.1 8.1 0.1 9.6 0.1 11.2 0.1 8.011.3n3 FA 1.3 0.1 1.4 0.1 0.7 0.1 1.0 0.1 0.8 0.1 0.61.5a SFA: saturated fatty acids; LC-SFA: long-chain SFA; UFA: unsaturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

1544 C. Lopez et al. / Food Research International 54 (2013) 15411552

ated using differential scanning calorimetry (DSC). (A) DSC thermograms recorded on cooling) DSC thermograms recorded on subsequent heating at 2 C/min from60 C to 60C (X-axis:

1545C. Lopez et al. / Food Research International 54 (2013) 15411552crystallization of at least two groups of TAG molecule as a function ofthe decrease in temperature. The initial temperature of crystallization(Tonset) was Tonset=16.71.3C. The sharp exotherm,with aminimumrecorded at about 16.4 0.8 C, corresponds to the rst liquid solidphase transition of TAG and could be attributed to the crystallizationof TAG containing long-chain saturated fatty acids (i.e. C18:0, C16:0).A second exotherm, broader than the rst one, was recorded fromT = 8 1.8 C. In some breast milk samples, a third exotherm wasrecorded around 15 C (arrows in Fig. 2A), and attributed to thecrystallization of TAG containing at least two unsaturated fatty acids.The presence or absence of this third exothermwas related to differencesin the unsaturated fatty acid amount in the milks expressed by themothers (from43 to 51.7%, Table 1). Crystallization of human TAG occursin awide range of temperatures andmature breastmilk fat is totally solidbelow30C. On subsequent heating, a broad endothermic massif wasrecorded, showing the melting of TAG on a wide range of temperatures.A rst endotherm spans from about 40 to 5 C with a maximumaround 5 C. Then a broad endotherm corresponding to the over-lapping of several endotherms ranged from 6.9 3.5 C to the nalmelting temperature of TAG (Toffset), Toffset=37.30.6 C.

The crystallization and melting of breast milk TAG occurs on a

Fig. 2. Thermal properties of anhydrous fat extracted from vemature breast milks investigat 2 C/min from 60C to60C (X-axis:35 to 45C). Arrows show a small exotherm. (B40 to 60 C). TAG: triacylglycerols (98% of milk lipids).wide range of temperatures, i.e. from30 to 37C. The supercooling,which corresponds to the difference between Tonset and Toffset wasToffset onset= 20.6 C. This supercooling is needed to initiate milkfat crystallization. The crystallization and melting properties ofwhole human milk TAG result from i) the high diversity of TAGmolecules due to fatty acids with various chain lengths and unsaturationleading to various melting points, and ii) to the polymorphism of milkTAG (Small, 1986). Our results are in agreement with recent literature,since similar Tonset, Toffset and melting proles of human TAG wererecently reported by Zou et al. (2013b).

3.3. Structural and thermal properties of TAGwithin breast milk fat globules

3.3.1. Size distribution of human milk fat globulesLipids are present in human milk in the form of spherical droplets

with polydisperse sizes (Fig. 3). TAG, that represent about 98% ofhuman milk lipids, have been labeled using Nile Red as hydrophobiclipid soluble uorescent probe (Fig. 3). The overlay of the imagestaken by light microscopy with DIC and by confocal microscopy usingNile Red showed that the TAG are exclusively located in the core ofhumanMFG, as previously reported (Lopez &Mnard, 2011).Whateverthe stage of lactation (from 2 to 7.5 months post-partum), the sizedistribution of mature human milk fat globules ranged from about 0.4to 15 m with a mean diameter of 4.9 0.4 m (Fig. 3). The surfacearea of human milk fat globules suspended in the surrounding aqueousphase was 2.0 0.1 m2/g of fat. These results are in accordance withprevious results reported in literature (Lopez & Mnard, 2011;Michalski, Briard, Michel, Tasson, & Poulain, 2005; Regg & Blanc,1981; Zou et al., 2012). The large surface area of milk fat globulesexposed to the lipolytic enzymes encountered during their passagethrough the digestive tract facilitates lipolysis of milk TAG andabsorption of the digestion products (Jensen et al., 1995).

3.3.2. Thermal properties of human milk fat globulesThe thermal properties of humanmilk fat globuleswere investigated

on cooling at |dT / dt| = 2 C/min from 60 to 5 C and subsequentheating at dT / dt = 2 C/min (Fig. 4). The experiments were notperformed for T b5 C to avoid ice formation in the milk samples.On cooling, at least two to three successive exothermal events havebeen recorded and attributed to the successive crystallization of TAGmolecules within human milk fat globules. The initial temperature ofcrystallizationwas Tonset=17.92.4C. Variations in the rst exothermwere recorded as a function of breast milks. This could be due to themechanisms of nucleation and crystal growth in emulsion, since in

conned systems such as milk fat globules crystallization occurs inindividual droplets. A second main exotherm was recorded for

Fig. 3. Structural characterization of fat globules in mature breast milks. Particle-sizedistributions of several mature breast milks determined using laser light scattering.Inset: Human milk fat globules characterized by light microscopy with differentialinterference contrast (DIC) and confocal laser scanning microscopy with the labeling oftriacylglycerols (TAGs) performed with Nile Red uorescent probe. The overlay of DICand confocal images shows the localization of TAG in the core of milk fat globules.

cann2C

1546 C. Lopez et al. / Food Research International 54 (2013) 15411552T b 5 C, with high variations in the enthalpy related to TAGcrystallization. The mechanisms of TAG crystallization in emulsion maybe affected by i) the size of fat globules, ii) the fatty acid composition,and iii) the molecules located at the TAG/water interface and able toinduce nucleation, that corresponds to the MFGM in the case of milkfat globules. The initial temperature of TAG crystallization was notsignicantly different between anhydrous TAG (Fig. 2) and fat globules(Fig. 4).

On subsequent heating, the DSC thermograms revealed a broad

Fig. 4.Thermal properties ofmature breastmilk fat globules investigated using differential saxis:5 to 45C). (B) Thermograms recordedon subsequent heating from5C to 60C atthe calorimeter). TAG: triacylglycerols (98% of milk lipids).endotherm. The nal melting temperature of TAG within human milkfat globules was Toffset = 39.8 1.5 C. The shape of the broadendotherm depended on the breast milk collected, and probably ondifferences in the fatty acid composition. The Toffset was above the in-body temperature of infants, i.e. 37 C.

3.3.3. Thermal properties and structural characteristics of human milk fatglobules upon storage at 4 C

The effect of the storage of breast milk in the refrigerator, at 4C, hasbeen investigated using the combination of biophysical techniques:confocal microscopy, DSC, and synchrotron radiation X-ray diffraction(XRD).

CLSM experiments performed at 4 C revealed the non-sphericaldistorted shape of human milk fat globules (Fig. 5). This may be dueto the presence of TAG crystals that have been formed in the core offat globules during storage at low temperature. TAG form needle orplatelet-shape crystals in emulsion droplets such as milk fat globules,as already observed (Lopez, 2011; Lopez et al., 2007; Walstra, 1967).Crystals dispersed in fat globules cannot grow larger than the globulediameter, but they can deform fat globules. Crystals can occulate in anetwork, giving the globule a certain rigidity (Mulder & Walstra,1974). Small fat crystals can be organized throughout the globule orcan be tangentially oriented at or near the boundary of the fat globule(Lopez et al., 2007; Walstra, 1967). Also, CLSM images showed thenon-homogeneous staining of TAG by the uorescent probe Nile Red(Fig. 5 D and E). This was interpreted as the partitioning of the probewith its preferential localization in the liquid phase of TAG. The solidstate of TAG molecules may avoid the integration of the hydrophobicuorescent probe.The thermal properties of TAG have been characterized by DSC, afterstorage of breast milks for 48 h at 4 C in the refrigerator. The DSCthermograms recordedwith themilks originating from severalmothersrevealed a broad multicomponent endotherm corresponding to themelting of TAG molecules within breast milks fat globules (Fig. 6). Themaximum of the endothermwas at 24.50.5C. The nal temperatureof TAG melting was Toffset=41.11.6 C. DSC experiments conrmedthat TAG molecules crystallized during the storage of breast milks at4 C.

ing calorimetry. (A) Thermograms recordedon cooling from60C to5C at 2C/min (X-/min (X-axis: 0 to 60C; theDSC signal recorded for tb0C corresponds to the launching ofThe structural organization of solid TAG molecules within humanmilk fat globules has been characterized at 4 C using high-uxsynchrotron XRD (Fig. 7) and on heating from 4 C to 60 C at 2 C/minusing the coupling of XRD and DSC (Fig. 8). Fig. 7 A shows the XRDpatterns recorded at small angles and Fig. 7 B theXRD patterns recordedat wide angles that correspond to the longitudinal stacking of TAGmolecules and to the lateral organization of the fatty acid chainsrespectively. The four XRD peaks recorded at small angles for q =0.150631, q=0.302381, q=0.453231 and q=0.549151

correspond to the formation of a lamellar structure with a double-chain length (2L) organization of TAG molecules with a thickness d=41.7 (noted 2L001; Miller index, h k l, with l=1, 2, 3 or 4). At wideangles, the four peaks with a major one around 4.6 were interpretedas the formation of a polymorphic form.

Themelting behavior of the TAG crystals formed upon storage at 4Cwas investigated on heating at 2 C/min from 4 C to 60 C (Fig. 8). Onheating, the XRD peaks recorded at wide angles simultaneouslydecreased in intensity (Fig. 8 A). For T N 43 C, the bump recorded atwide angles (q=1.41) corresponds to the scattering signal of TAGmolecules in their liquid state. At small angles, the XRD peaks relatedto the 2L (41.7) structure simultaneously decreased in intensity untiltheir disappearance for T N 43 C (Fig. 8 B). The thickness of the 2Lstructure did not evolved signicantly during its melting. The absenceof molecular rearrangements and polymorphic transition until nalmelting of TAG demonstrates the stability of the crystalline structureformed. The DSC signal recorded simultaneously to XRD experimentsshow a broad endotherm (Fig. 8 C), corresponding to the meltingof TAG molecules. The nal melting point of TAG determined usingDSC, Toffset = 43 C, corresponds to the disappearance of the XRD

1547C. Lopez et al. / Food Research International 54 (2013) 15411552peaks recorded at both small and wide angles. At the temperature ofmilk digestion by infants, i.e. Tbody = 37 C, breast milk fat globulesare partially crystallized. Indeed, a liquid phase of TAG coexistswith solid TAG crystals organized in a 2L (41.7 ) lamellarstructure.

Storage of breast milk at room temperature (i.e. 19 C) leads to theformation of similar TAG crystals than storage at 4 C: 2L (41.7 )(Fig. 9). The bump recorded at q= 0.265 1 (23.7 ) corresponds tothe scattering of TAG in their liquid state. The relative intensity of thescattering signal related to the liquid phase was higher at 20 C ascompared to 4C (Fig. 8)meaning a higher amount of liquid TAGwithinbreast milk fat globules at 20 C.

Fig. 5.Microstructure of fat globules upon storage of breast milk at 4 C for 48h characterized bymicroscopy with the labeling of triacylglycerols (TAG) performed with Nile Red uorescent pro(D, E) Confocal images showing the non-homogeneous distribution of Nile Red in the TAG corStorage of breast milk for 48 h at 4 C (and at room temperature)leads to the crystallization of TAG within milk fat globules with theformation of crystals, which is the most thermodynamically stablepolymorphic form of TAG with a compact organization of the fattyacid chains. The physical properties of TAG molecules depend on theirfatty acid composition and on the position of the fatty acids on theglycerol backbone. According to literature, the long spacings of TAGorganized in crystals vary linearly according to the relation: d()=2.27 n+4.11 (n is the number of atoms of carbon in the hydrocarbonchain), which corresponds to an angle of 63 (Small, 1986). Hence,we calculated that the long spacings of 41.7 correspond to thelongitudinal organization of TAG with 16.6 atoms of carbon in one

light microscopywith differential interference contrast (DIC) and confocal laser scanningbe. (A, B, C) Overlay of DIC and confocal images showing the deformation of fat globules.e of fat globules. Scale bar= 10 m.

Fig. 6.Thermal properties ofmature breastmilk fat globules investigated after 48h storageat 4 C using differential scanning calorimetry. Thermograms recorded on heating of thesamples from5 C to 60 C at 2 C/min (X-axis: 0 to 60 C; the DSC signal recorded fortb0C corresponds to the launching of the calorimeter). Thenal temperatures ofmeltingare indicated in the gure.

Fig. 7. Crystalline structures formed within milk fat globules after 48 h storage at 4 Cidentied by high-ux synchrotron X-ray diffraction (XRD). (A) Small-angle XRDpatterns; the longitudinal double chain length (2L) is labeled with the reections (Millerindex, h k l, with l=1, 2, 3, 4). (B) Wide-angle XRD patterns. q: scattering vector.

1548 C. Lopez et al. / Food Research International 54 (2013) 15411552layer. Then, according to the fatty acid composition of breast milk(Table 1), we emit the hypothesis that the crystals are formed byTAG containing palmitic acid (P; n = 16), and fatty acids with n N 16such as stearic acid (S; n=18) and oleic acid (O; n=18 and one cis-unsaturation). The complex TAG composition of human milk may leadto the formation of compound crystals, i.e. crystals containing morethan one species of TAG that can accommodate in 2L (41.7) crystals.The trisaturated TAG containing P and S are present in humanmilkwithlow amounts: SPP (0.20%), PPP (0.38%) and PPM (1.20%) (Zou et al.,2013a). The main breast milk TAG containing two long-chain saturatedfatty acids (P, S) and one long-chainmonounsaturated fatty acid (O) arePPO and SPO (12.14% and 2.29% of TAG, respectively; Zou et al., 2013a).Hence, it is highly probable that PPO, SPO and trisaturated TAG are themain component of the crystals formed upon storage of breast milk at4 C. The double chain length organization of the crystals means thatthe saturated (P, S) and cis-unsaturated (O) chains are packed in thesame leaet (Fig. 10); the cis-unsaturated fatty acid chain (i.e. oleicacid) was not segregated in a leaet to form 3L structure. This is inagreement with previous studies describing the polymorphic forms ofpure PPO molecules (Minato, Ueno, Smith, Amemiya, & Sato, 1997).However, structural parameters of PPO in a complex mixture of TAGsuch as human milk fat have never been reported before. Moreover, thecrystallization properties of PPO dispersed in emulsion droplets hasnever been studied. In a previous study, Lopez et al. (2002) showedthat the storage of bovinemilk fat globules at 4C leads to the coexistenceof 2L (40.5) and 3L (54.2) organizations associatedwith,2,1 and packings of the chains. The absence of short-chain fatty acids in humanmilk and the specic internal structure of TAG with most of C16:0 in thesn-2 position has led to the formation of 2L (41.7 ) crystals uponstorage of breast milk at 4 C. This study conrms that the chemicalcomposition of TAG governs their crystallization properties.

3.4. General discussion: potential role of the solid TAG phase formed withinhuman milk fat globules

Breast milk has been optimized through millennia of evolution to bethe best source of nutrients and non-nutrient components for theoptimal growth and development of infants. The milk secreted by themammary glands of the mothers is supposed to be transferred directlyto the infant, at the in-body temperature (Tbody = 37 C). However,mothers can express their milk by pumping and store breast milk invarious conditions including storage at low temperatures, e.g. in therefrigerator for several days and in the freezer for months. In thisstudy, we investigated the effect of cooling breast milk at T b Tbody onthe physical properties of fat and revealed that TAGmolecules crystallizewithin human milk fat globules upon storage at 4 C. Moreover, weshowed that the nal melting temperature of these TAG crystals isabove the in-body temperature (i.e. ToffsetN Tbody). The presence of solidTAG within milk fat globules can have several consequences that needto be further discussed.

The nal melting point of anhydrous human milk fat was Toffset =37.30.6 C, which is close to in-body temperature and in agreementwith literature (Zou et al., 2013b). The nal melting point of the sameTAG dispersed in milk fat globules (i.e. natural vehicles of TAG in breastmilk) was above in-body temperature. Moreover, we showed that thepolymorphism of TAG molecules affects the nal melting point ofhuman milk fat globules since it was Toffset=39.81.5 C after coolingfrom themelt (i.e. 60C) and Toffset=41.11.6C after 48hour storageat 4C. This difference of Toffset=1.3C is interpreted as differences inthe polymorphic forms of high-melting point TAG crystals ( versus or versus ; the most stable polymorphic form having the highestmelting point; Small, 1986).

Storage of breast milk for 48h at 4 C induced the formation of 2L(41.7 ) crystals within fat globules, as identied using synchrotronradiation XRD at both small and wide angles. The phase transition of

TAG molecules from the liquid state to polymorphic form may lead

Fig. 8. Structural and thermal evolution of TAGwithin breast milk fat globules on heating at 2 C/min from 4 C to 65 C, after storage for 48h at 4 C. (A) Three-dimensional plots of wide-angle X-ray diffraction patterns recorded during heating. (B) Evolution of the maximal intensity of the main small-angle diffraction peak recorded which corresponds to long spacings(100% corresponds to the strongest peak); inset: Three-dimensional plots of the small-angle X-ray diffraction peak. (C) Differential scanning calorimetry curve recorded simultaneouslyfrom the same sample.

1549C. Lopez et al. / Food Research International 54 (2013) 15411552

to a decrease in the volume of fat globules since the volume occupied bya methylene group is 303 for liquid TAG and 23.53 for the sub-cell(Small, 1986). Hence, crystallization of TAG during storage of breast

changes in volume affect the properties of the MFGM, its protectivefunction on the gastrointestinal tract of infants is uncertain (Hamoshet al., 1999; Mulder & Walstra, 1974). The MFGM is a natural barrier

Fig. 9.Crystalline structures formedwithinmilk fat globules after 48h storage at 19C identiedbyhigh-ux synchrotron X-ray diffraction (XRD) at small andwide angles. At small angles,the longitudinal double chain length (2L) is labeled with the reections (Miller index, h k l, with l=1, 2, 3, 4). q: scattering vector.

1550 C. Lopez et al. / Food Research International 54 (2013) 15411552milk at 4 C can lead to a decrease in the volume and to contraction offat globules, as previously discussed for the storage of bovine milk at4 C (Lopez et al., 2002). By contraction during solidication of TAGand also probably from the irregular shape of the TAG crystals, partlysolidied milk fat globules were non-spherical. Whether these localFig. 10. Schematic representation of the solid phase formed by triacylglycerols (TAG) in the cpolymorphic form with a double chain length (2L 41.7) longitudinal organization of TAG mothat protects TAG from lipolysis. However, it is possible that thecrystallization of TAG molecules favors the accessibility of the humanbile salt-stimulated lipase (BSSL; lipase secreted in breast milk) to theTAG core of fat globules and then increases lipolysis upon storage ofbreast milk in the refrigerator (Bertino et al., 2013; Neville, 1995). Theore of breast milk fat globules upon storage at 4 C for 48 h. Proposed structure of the lecules as seen in the ca projection.

The authors thank the volunteermothers for their donations of breast

own milk reduces fat absorption and growth in preterm infants. Acta Paediatrica,

1551C. Lopez et al. / Food Research International 54 (2013) 15411552alteration of the MFGM as a result of solid TAG constraints and theformation of diacylglycerols, monoacylglycerols and free fatty acidsduring storage of breast milk in the refrigerator could impact on themechanisms of TAG digestion in newborns (Lindquist & Hernell, 2010).

Humanmilk fat globules are themain source of energy and essentialnutrients for breast-fed infants. TAG molecules constitute half ofthe energy content in human milk. Hence, efcient hydrolysis andabsorption of TAG is important, not only for energy utilization but alsofor optimal growth (Lindquist & Hernell, 2010). Jensen (1999) reportedthat fat globules remain liquid in the stomach and small intestineof infants for an optimal hydrolysis by the digestive enzymes and anoptimal absorption. The effect of storage of breast milk at low tem-perature on the solid or liquid state of TAG within fat globules was notdocumented. In this study, we revealed that the nal melting point of crystals formed upon storage of breast milk at 4 C is above the in-body temperature (Toffset N Tbody) and then that TAG are partially solidat the temperature of milk digestion by infants. These data are totallynew. To be assimilated by the infant, TAG need to be hydrolysed bythe digestive enzymes and solubilized. Digestion of milk fat globulesdepends on the consecutive action of several lipases: BSSL, gastric lipasein the stomach and pancreatic lipaserelated proteins in the duodenumof infants (Lindquist & Hernell, 2010). The presence of solid TAGmolecules in the core of fat globules after storage at 4 C raises thequestion of the action of digestive lipolytic enzymes on a solid substrate,on their solubilization and then on the absorption and metabolism oflipids. Palmitic acid C16:0, that is mainly located in the sn-2 positionof TAG in human milk, is an important fatty acid for breast-fed infantnutrition and development (Jensen, 1999; Lindquist & Hernell, 2010).The crystallization of TAG molecules containing C16:0 (i.e. PPO, SPO,PPP, PPM, SPP) could limit their hydrolysis by the digestive lipolyticenzymes and then limit the absorption of sn-2 diacylglycerols containingC16:0 with nutritional consequences in the infant that remain to beinvestigated. Also, TAG crystals located at the periphery of fat globulescould prevent the accessibility of lipases to the liquid TAG located in thecenter of fat globules. Hence, the solid fat phase in the gastro-intestinaltract of newborns could decrease the rate of TAG hydrolysis, the kineticof fatty acid absorption and bioavailability. The inuence of the physicalstate of TAG and particularly the proportion of solid fat on lipid digestionand absorption remains poorly documented (Asselin et al., 2004; Berry,Miller, & Sanders, 2007; Bonnaire et al., 2008; Golding et al., 2011;McClements, 2012). Hence, whether the physical state of TAG (i.e. extentof solid fat content) has an inuence on the digestion and metabolism oflipids in infants remains poorly known. Authors reported that thecoefcient of fat absorption in infants decreases with increasing chainlength of a fatty acid and increases with increasing number of doublebonds of the fatty acid (unpublished data from Andersson, Savman,Blackberg, & Hernell, 2007 cited in Lindquist & Hernell, 2010). Thiscould be related to the high melting point of long chain saturated fattyacids that are solid in the digestive tract of infants.

The role played by solid TAG in the gastro intestinal tract of infantsshould be further considered. We hypothesize that crystallization ofhuman milk TAG within fat globules upon storage of breast milk at 4 Ccould decrease the amount of utilizable fat for the recipient infant, inthe case breast milk is consumed at T b Toffset of TAG crystals. Aftercollection of breast milk and its storage in the refrigerator, thepreparation of the breast milk to be fed to the infant by bottle couldhave a nutritional and physiological importance. Indeed, for Tb Toffset ofTAGcrystals, some solid TAGwill remain in breastmilk,whereas for TNT-offset of TAG crystals, whole TAG will be liquid. Warming breast milk atT4550 C N Toffset of TAG crystals formed upon storage in the refrigeratoris then important to ensure breast milk TAG digestibility and absorption.

4. Conclusion

This study revealed the formation of a solid TAG phase within fat

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Bitman, J., Wood, D. L., Hamosh, M., Hamosh, P., & Mehta, N. R. (1983). Comparison of thelipid composition of breast milk frommothers of term and preterm infants. AmericanJournal of Clinical Nutrition, 38, 300312.

Blanton, T. N., Barnes, C. L., & Lelental, M. (2000). Preparation of silver behenate coatingsto provide low- tomid-angle diffraction calibration. Journal of Applied Crystallography,33, 172173.

Bonnaire, L., Sandra, S., Helgason, T., Decker, E. A., Weiss, J., & McClements, D. J. (2008).Inuence of lipid physical state on the in vitro digestibility of emulsied lipids.Journal of Agricultural and Food Chemistry, 56, 37913797.

Bugeat, S., Briard-Bion, V., Prez, J., Pradel, P., Martin, B., Lesieur, S., et al. (2011).Enrichment in unsaturated fatty acids and emulsion droplet size affect thecrystallization behaviour of milk triacylglycerols upon storage at 4 C. Foodmilk samples. The authors thank synchrotron SOLEIL for allocatingbeamtime on the SWING beamline (proposal 20080314; C. Lopez), aswell as A. Bulon (INRA, BIA, Nantes, France) and all the members ofthe Scientic Users Committee of INRA.

Contributors

Christelle Lopez: Coordination of the study; writing of the paper;laser light scattering experiments; microscopy characterizations; DSCand DSC/XRD experiments.

Valrie Briard-Bion: Fatty acid analysis.Claudie Bourgaux: DSC/XRD experiments.Javier Perez: DSC/XRD experiments (responsible for the synchrotron

beamline).The authors declare no conict of interest.

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Solid triacylglycerols within human fat globules: crystals with amelting point above in-body temperature of infants, formed uponstorage of breast milk at low temperature1. Introduction2. Materials and methods2.1. Breast milk samples and anhydrous fat2.2. Fat globule size measurements2.3. Confocal laser scanning microscopy2.4. Fatty acid composition2.5. Differential scanning calorimetry (DSC) experiments2.6. Time-resolved and temperature-controlled synchrotron X-ray diffraction (XRD) experiments and XRD/DSC coupling

3. Results and discussion3.1. Fatty acid composition of mature breast milks3.2. Thermal properties of anhydrous human milk fat3.3. Structural and thermal properties of TAG within breast milk fat globules3.3.1. Size distribution of human milk fat globules3.3.2. Thermal properties of human milk fat globules3.3.3. Thermal properties and structural characteristics of human milk fat globules upon storage at 4C

3.4. General discussion: potential role of the solid TAG phase formed within human milk fat globules

4. ConclusionAcknowledgmentsReferences