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Lait 87 (2007) 459–480 Available online at:c© INRA, EDP Sciences, 2007 www.lelait-journal.orgDOI: 10.1051/lait:2007018

Original article

Coupling of time-resolved synchrotron X-raydiffraction and DSC to elucidate

the crystallisation properties and polymorphismof triglycerides in milk fat globules

Christelle Lopeza,b*, Claudie Bourgauxb,c, Pierre Lesieurc,d,Michel Ollivonb

a UMR1253 Science et Technologie du Lait et de l’Œuf, INRA-Agrocampus Rennes,65 rue de Saint-Brieuc, 35042 Rennes Cedex, France

b UMR8612 CNRS Université Paris-Sud, Physico-Chimie des Systèmes Polyphasés,92296 Châtenay-Malabry, France

c Laboratoire pour l’Utilisation du Rayonnement Électromagnétique (LURE),91898 Orsay, France

d Physico-Chimie des colloïdes, Université Henri Poincaré, UMR7567, BP 239,54506 Vandœuvre-lès-Nancy, France

Abstract – Crystallisation properties and polymorphism of triacylglycerols (TG) in natural milk fatglobules are studied with an apparatus permitting the coupling of time-resolved synchrotron X-raydiffraction (XRD) and high-sensitivity differential scanning calorimetry (DSC) for cooling rates0.1 < Rcooling < 1000 ◦C·min−1. We showed that the fat phase of dairy products displays a complexpolymorphism. For the first time, six different types of crystals were identified, several of them incoexistence, and their time- and temperature-dependent evolutions were quantitatively monitored.They correspond to lamellar structures with double-chain length (2L: 40.5–48 Å) and triple-chainlength (3L: 54–72 Å) organisations of TG. Depending on the cooling rate, at least five crystallinesubcell species were observed at wide angles: α and sub-α, two β’ and one β. All these crystallinestructures coexist with a liquid phase even at low temperatures. Thermal events recorded by DSCwere related to the structural information on the organisation of TG obtained by XRD. Moreover,we showed that crystallisation in fat globules induces a higher disorder and a smaller size of crystalsthan in bulk, i.e. in anhydrous milk fat. Furthermore, XRD permitted the identification of differentcrystallisation behaviour in natural milk fat globules with different sizes, which could be implicatedin the manufacture of dairy products involving tempering periods in the technological process (e.g.butter, ice cream, whipped products). The increased knowledge of milk fat crystallisation in water-dispersed and highly complex food products might have a certain value for technical applicationsas well as for the development of new processes and dairy products.

crystal / phase transition / triacylglycerol / emulsion droplet / fat globule size

摘摘摘要要要 –时时时间间间分分分辨辨辨的的的X射射射线线线衍衍衍射射射和和和差差差示示示量量量热热热扫扫扫描描描法法法研研研究究究乳乳乳脂脂脂肪肪肪球球球中中中三三三酸酸酸甘甘甘油油油酯酯酯的的的晶晶晶体体体性性性质质质和和和多多多晶晶晶性性性。。。本文采用时间分辨的X射线衍射仪 (XRD)和高灵敏度的差示量热扫描仪(DSC)在冷却速率为 0.1 < Rcooling < 1000 ◦C·min−1 时,研究了天然乳脂肪球中三酸甘油酯 (TG)晶体特性和多晶性。实验证明乳制品中的脂肪相具有复杂的多晶性。首次鉴定出 6种不同的晶型,他们中大多数是共存的,并且定量地测定了这六种晶型的时间和温度之间的变化关系。他们对应的层状结构是具有双倍链长(2L: 40.5–48 Å)和三倍链长 (3L: 54–72 Å)的 TG组织。

* Corresponding author (通讯作者): [email protected]

Article published by EDP Sciences and available at http://www.lelait-journal.org or http://dx.doi.org/10.1051/lait:2007018

http://www.edpsciences.org

http://www.lelait-journal.org

http://dx.doi.org/10.1051/lait:2007018

460 C. Lopez et al.

根据冷却速率,在一个很宽的范围内至少观察到 5 个亚晶胞结构, 分别为α、亚-α、2个 β′

和1个 β型结构。即使在较低的温度下,这些晶型结构也是与液态相共存的。DSC的热分析结果与 XRD观察到的 TG组织结构特性是一致的。此外,我们还证明了脂肪球中的脂肪晶体与游离脂肪 (如无水脂肪)相比, 前者处于高度的无序状态,并且晶体的尺寸较小。根据XRD分析证明了不同大小的天然乳脂肪球具有不同的晶体性质，这种性质有可能与乳制品(如奶油、冰淇淋、搅打乳制品)加工过程中所涉及到老化工艺有直接的关系。了解乳脂肪晶体在水分散的和复杂的食品体系中的结晶特性将有助于解决生产实际问题和开发新的加工技术及新的乳制品。

晶晶晶体体体 /相相相转转转移移移 /三三三酸酸酸甘甘甘油油油酯酯酯 /乳乳乳状状状液液液滴滴滴 /脂脂脂肪肪肪球球球大大大小小小

Résumé – Couplage de la DSC et de la diffraction des rayons X résolue en temps en utili-sant le rayonnement synchrotron pour élucider les propriétés de cristallisation et le polymor-phisme des triglycérides dans les globules gras du lait. Les propriétés de cristallisation et lepolymorphisme des triglycérides (TG) dans les globules gras naturels du lait sont étudiés avec unappareil permettant le couplage de la diffraction des rayons X (DRX) résolue en temps et de lamicrocalorimétrie différentielle (DSC), pour des vitesses de refroidissement comprises entre 0,1 et1000 ◦C·min−1. Nous avons montré que la phase lipidique des produits laitiers possède un polymor-phisme complexe. Pour la première fois, six types différents de cristaux ont été identifiés, plusieursd’entre eux étant en coexistence, et leurs évolutions ont été caractérisées de manière quantitative,en fonction du temps et de la température. Elles correspondent à des structures lamellaires avecdes structures à deux longueurs de chaîne (2L : 40,5–48 Å) et à trois longueurs de chaîne (3L :54–72 Å). En fonction des vitesses de refroidissement, au moins cinq sous-cellules cristallines ontété identifiées aux grands angles : α, sub-α, deux β′ et β. Toutes ces structures cristallines coexistentavec une phase liquide, même à basse température. Les évènements thermiques enregistrés par DSCont été reliés aux informations structurales de l’organisation des TG obtenues par DRX. Nous avonsmontré également que la cristallisation dans les globules gras induit un plus grand désordre et uneplus petite taille des cristaux qu’en milieu anhydre. De plus, la DRX a permis d’identifier différentscomportements de cristallisation dans les globules gras de différentes tailles, qui pourraient inter-venir lors de la fabrication de produits laitiers nécessitant des périodes de tempérage (par exemplele beurre, la crème glacée, les produits foisonnés). L’augmentation des connaissances sur la cris-tallisation de la matière grasse du lait dans les systèmes dispersés et dans les produits alimentairescomplexes contribuera aux innovations technologiques ainsi qu’au développement de nouveauxproduits.

cristal / transition de phase / triglycéride / gouttelette d’émulsion / taille des globules gras

1. INTRODUCTION

Triacylglycerols (TG), which are themain constituents of natural fats, deter-mine the physical and thermal properties ofhigh-fat products. Moreover, the fat phaseof food products is sometimes partiallycrystallised at room and storage (4–7 ◦C)temperatures, as is the case for milk anddairy products.

Milk fat is an essential nutriment con-sumed in many food products, in whichit is frequently found dispersed as smalldroplets called milk fat globules, e.g. inmilk, cream and cheeses. Crystallisation of

TG in milk fat globules is of prime im-portance because it affects many propertiessuch as (i) the rheological properties, (ii)the resistance of fat globules to disruptionand then to coalescence, (iii) the suscepti-bility of globules to churning, (iv) the sta-bility of whipped cream and (v) the consis-tency and mouth feel of high-fat products.Thus, it is important to understand betterthe physical properties of fat globules, e.g.their thermal and crystallographic proper-ties, for industrial applications and to im-prove the quality of food products.

Milk fat globules have a diame-ter ranging from 0.02 to 15 μm with

Crystallisation of triglycerides in emulsion 461

−50 −40 −30 −20 −10 0 10 20 30 40 50Temperature (°C)

HF

End

o> LMPF

MMPF

HMPF

Figure 1. Thermal properties of anhydrous milk fat. Crystallisation and melting curves recordedupon cooling at 2 ◦C·min−1 and subsequent heating at 2 ◦C·min−1 using differential scanningcalorimetry (DSC). LMPF: low melting point fraction; MMPF: middle melting point fraction;HMPF: high melting point fraction.

a volume-weighted diameter of about4 μm [9]. Natural milk fat globules, whichare enveloped by a biological membrane(the milk fat globule membrane, MFGM),are mainly composed of TG (∼98% ofmilk lipids), which are esters of fattyacids and glycerol. More than 400 fattyacids have been identified in milk, witha wide diversity of chain lengths, num-ber of unsaturation, branching and positionon the glycerol. More than 200 individ-ual molecular species of even-numberedTG have been quantified [4]. Furthermore,it is well known that milk fat compo-sition changes with cow feeding, seasonand stage of lactation. Thus, milk fat isundoubtedly the most complex fat foundin nature. The consequence of this TGand fatty acid composition is that anhy-drous milk fat (AMF), which is the fatisolated from butter, has a broad meltingrange from −40 ◦C to +40 ◦C (Fig. 1)and no true melting point, like pure com-pounds. Therefore, at intermediate temper-atures and at equilibrium (e.g. room andfridge temperatures) milk fat is a mix-ture of crystals and oil. Moreover, this

fat-composition-related complexity is dra-matically enhanced by the existence of apolymorphism of monotropic type for eachTG [22, 25]. As observed for most of thelipids, each TG of milk fat can exhibitseveral crystalline forms, the occurrenceof which strongly depends on its thermalhistory. Each polymorphic form is charac-terised by its own melting point. Further-more, the fact that TG mixtures also ex-hibit multiple melting points, depending ontheir composition, renders the overall melt-ing behaviour of milk fat even more com-plex.

TG polymorphism relates to the abil-ity of molecules to arrange themselveswithin a crystal lattice in a number ofdifferent ways of lateral packing of thefatty acid chains and of longitudinal stack-ing of molecules in lamellar structures(Fig. 2) [6]. Thus, pure TG and mix-tures of TG can adopt several crystallinearrangements. Transitions between thesepolymorphic forms are mostly irreversible;monotropism implies that they are onlypossible via a liquid and when leading tothe formation of more stable species [25].

462 C. Lopez et al.

O ⊥⊥ T //Hexagonalas

bs

2.54 Å2.54 Å Endview

(B)Lateral Organisation of Fatty Acid chainsForm β of trilaurin

δ

(C)

Saturated FA

Unsaturated FA

Saturated FA

Saturated FA withsimilar chain length

Short-chain FA

Saturated FA

Saturated FA

3L : Triple-chain length lamellar structure (55-75 Å)

2L : Double-chain length lamellar structure (35-50 Å)2 nm

Longitudinal Organisation of TG Organisation of TG moleculesmolecules

(A) Lamellar structure

Figure 2. Main types of TG packings. (A) Lamellar structure formed by triacylglycerol (TG)molecules in the solid state: example of the β form of trilaurin. (B) Top: the stable conforma-tion of the hydrocarbon chains of saturated fatty acid (FA) is a planar zigzag shown here as a 3Dview along its main axis; bottom: three main types of chain lateral packings (only carbon atoms aredrawn, hydrogen atoms are not drawn): hexagonal, orthorhombic perpendicular (O⊥) and triclinicparallel (T//) subcells to which α, β’ and β polymorphic forms correspond. (C) Two main types ofTG chain longitudinal stacking (fatty acids are drawn as straight lines).

The three main polymorphic forms fre-quently observed for the lateral pack-ing of fatty acid chains correspond todifferent subcells that have been describedin detail [22, 25]: hexagonal (α form), or-thorhombic perpendicular (β’ form) andtriclinic parallel (β form) (Fig. 2B). Thedensity, enthalpy of fusion, melting pointand stability increase in the order α, β’ and

β, according to the monotropic characterof the polymorphism. In the α form, thepacking of chains is not very tight and thechains have considerable rotational free-dom, whereas in the β form, the chainsare very densely packed. TG crystals aremade by the stacking of TG molecule lay-ers, the thickness of which depends onthe length and unsaturation of the fatty


acid chains, and their angle of tilt with re-spect to the basal planes formed by themethyl end groups of the TG (Fig. 2C). Thelongitudinal organisations of TG in lamel-lar structures are primarily related to thenumber of chains stacked one on top of theother in the main crystalline cell. For TG,this number frequently takes a value of 2 or3 and corresponds to the stacking of dou-ble (2L) or triple (3L) chain length lamellarstructures, leading to distances in the range35–50 Å and 55–75 Å, respectively [6,25].Roughly, 3L forms are usually related tolow-melting, long-chain monounsaturatedand/or mixed long- and short-chain TG,whereas 2L forms are generated mostlyby similar long-chain, high-melting, trisat-urated TG [25].

Crystallisation in milk fat globules hasbeen investigated by different techniques.Freeze-fracture electron microscopy hasmade possible the study of fats and foodsystems such as milk, cream and but-ter [24, 26]. Polarised light microscopypermitted the classification of fat glob-ules into four main types as a functionof the crystal habit [14, 28]. These stud-ies gave an insight into the orientationand the size of the crystals at a micro-scopic level, but little information exists ata molecular level on the organisation of TGmolecules in the crystals formed within fatglobules. The techniques most frequentlyused for the study of the thermal and crys-tallographic properties of TG are differen-tial scanning calorimetry (DSC) and X-raydiffraction (XRD), respectively.

DSC studies have given an insight intothe thermodynamics of fat phase transi-tion in bulk and in emulsions. The authorsthat studied anhydrous milk fat by DSCobserved that it crystallises and melts inseveral steps (Fig. 1). A typical meltingcurve of AMF shows three endothermicpeaks, corresponding to low melting point(LMP), medium melting point (MMP) andhigh melting point (HMP) fractions [27].These peaks correspond to large groups of

TG that melt separately and behave likesolid solutions. The occurrence of numer-ous thermal transitions during DSC exper-iments, the partial overlapping of the melt-ing peaks, their respective enthalpies andtemperatures depend strongly on the ther-mal treatments (e.g. heating and coolingrates, tempering) and on the entire thermalhistory of the sample [1, 22]. The complexDSC recordings are difficult to interpret.Thus, DSC experiments need to be coupledwith other techniques.

XRD is an essential tool for elucidat-ing the molecular packing of moleculesin the solid state and polymorphism ofpure TG and complex fats, since it pro-vides structural information on the organ-isation of molecules, and it complementsDSC. X-rays are the ideal direct probe todetermine the internal structure of crys-tals, since they provide information on therepetitive patterns of the electron density ofthe array of atoms. The atoms that consti-tute the TG molecules inside a perfect crys-tal are arranged periodically in planes at arepetitive distance, d (Fig. 3). As a beamof X-rays comes to the crystal at an an-gle, θ, the X-rays of wavelength λ are re-flected by the planes at that same angle θ.The optical path length required to produceconstructive interference is nλ, with inte-gral values of n, providing specific crys-tal symmetry allows it. The equation thatdetermines the angle at which constructiveinterference occurs is known as Bragg’slaw: 2dsinθ = nλ [5,25]. The actual diffrac-tion measurement during an experiment isthe one formed between the incident beamand the reflected beam, which is denotedby 2θ. It is often convenient to use thescattering vector q instead of the scatter-ing angle 2θ since the former is indepen-dent of the X-ray wavelength. They are re-lated as follows: q = (4π/nλ) sinθ. Thus,q = 2π/d.

The two levels of organisation, e.g. thelateral packing of the fatty acid chains andthe longitudinal stacking of TG molecules

464 C. Lopez et al.

2 d sinθθ = n λ

2θ

d

X-rays

θ

λ ~ 1.5 Å

θ

Figure 3. Demonstration of Bragg’s law of diffraction: 2dsinθ = nλ (d in Å is the repetitive distancebetween the planes arranged periodically; θ in degrees is the angle of incidence of X-ray relative tothe crystalline plane; λ in Å is the X-ray wavelength; the integral values of n are required to produceconstructive interference nλ).

in lamellae formed in the solid state(Fig. 2), are easily identifiable from XRDpatterns recorded at wide and small angles,respectively. The thickness of the lamel-lar structures, which can be measured byXRD at small angles (0◦ < θ < 5◦),corresponds to TG longitudinal stackingcalled TG long spacings. From the mea-surement of d (Å) and with knowledge ofthe fatty acid composition (chain lengthand unsaturation), it is possible to deduceif the stackings correspond to 2L or 3L or-ganisations (Fig. 2C). The cross-sectionalpackings of the aliphatic chains are charac-terised by specific short spacings, indepen-dent of chain length and observable at wideangles in the range 8.5◦ < θ < 13◦. Shortspacings are widely used for identifyingthe various crystalline subcells character-ising the polymorphic forms (Fig. 2B). Asingle line around 4.15 Å characterises theα form (hexagonal subcell), and a strongline at 4.6 Å among other sharp lines iden-tifies the β form (triclinic parallel subcell),while the β′ form (orthorhombic perpen-dicular subcell) frequently shows associa-

tion of two lines, among which one is about4.2 Å and the other around 3.8 Å.

Recent use of synchrotron radiation,which provides X-ray flux 103 to 106 timesmore intense than that generated by usualX-ray sources, permits recordings to beperformed within a few seconds or mil-liseconds. Thus, direct continuous record-ings can be performed as a functionof time (XRDt) or temperature (XRDT).Moreover, synchrotron radiation permitsthe study of the organisation of TG inwater-dispersed systems such as emulsionsand food products and to quantitativelymonitor phase changes within emulsiondroplets. This is especially interesting torelate the textural and rheological proper-ties of milk fat, and of high-fat food prod-ucts, to the thermal and crystallographicproperties of TG. Therefore, the functionof milk fat in many food products can-not be understood without knowing bothits composition and physical properties, aswell as those of their respective dependen-cies.


Determination of the thermal and crys-tallographic properties of TG molecules inthe dispersed state, e.g. in milk fat glob-ules, is much more challenging than forfat in bulk, e.g. anhydrous milk fat, sincethe presence of numerous compounds (e.g.casein micelles, minerals, lactose and wa-ter) not involved in the TG organisationwill necessarily decrease the intensity ofthe signal recorded by both DSC and XRDtechniques, by a simple dilution effect.Overcoming the difficulties associated withmilk fat globule examination requires pow-erful X-ray sources such as those availableat synchrotron laboratories.

The objective of this paper is to pro-vide an overview of the possibilities ofinvestigation offered by the coupling ofhigh-sensitivity DSC and time-resolvedsynchrotron radiation XRD. Selected ex-amples illustrate the study of the thermaland crystallographic properties of milk fatwithin the globules, the evolution of organ-isation of milk fat TG in the solid state andtheir polymorphism as a function of tem-perature and time.

2. MATERIALS AND METHODS

2.1. Samples

Concentrated creams (fat content = 400to 800 g·kg−1) were obtained by industrialskimming of whole milk. Anhydrous milkfat (AMF) was extracted from the creamsas detailed in Lopez et al. [15]. Naturalmilk fat globules with different sizes wereselected from the same raw whole milk byusing a novel microfiltration process [21].

2.2. Methods

2.2.1. Coupled XRD and DSCmeasurements

Experiments were performed using atechnique allowing the coupling of time-resolved synchrotron X-ray diffraction as

a function of temperature (XRDT) or time(XRDt) and high-sensitivity differentialscanning calorimetry (DSC) in the sameapparatus from the same sample. This ap-paratus, called Microcalix, was recentlydescribed in Ollivon et al. [23].

This set-up was used on the D22 andD24 benches of the DCI synchrotronof LURE (Laboratoire pour l’utilisationdu rayonnement électromagnétique, Orsay,France). On the D22 bench (λ = 1.5498 Å),the XRD data were recorded simulta-neously at small and wide angles withtwo position-sensitive gas linear detec-tors (1024 and 512 channels), which wereplaced about 1 770 mm and 300 mm fromthe sample, respectively (Fig. 4). On theD24 bench (λ = 1.489 Å), a single detectorcan be placed at 300 mm or 900 mm (q =0–0.55 Å−1). The small-angle detector al-lows the characterisation of the longitudi-nal organisation of TG molecules, whereasthe wide-angle detector permits the identi-fication of the chain packing (Fig. 2). Thechannels of the detectors were calibrated toexpress the XRD data in scattering vector q(q = 4π sin θ/λ; q in Å−1, θ in degrees isthe angle of incidence of the X-ray rela-tive to the crystalline plane, λ in Å is theX-ray wavelength) [5]. The crystalline 2Lβ form of high-purity tristearin was used asa reference for the wide-angle channel toscattering vector q calibration of the detec-tor (4.59, 3.85, 3.70 ± 0.01 Å) [22]. Silverbehenate, which is characterised by a longspacing of 58.380 ± 0.001 Å, was used tocalibrate the small-angle detector [3]. Thecalorimeter coupled to XRD was calibratedwith lauric acid.

XRD data and DSC measurements weresynchronously collected versus time bya single microcomputer. A Visual Basic(Microsoft, Redmond, USA) program wasspecially written to ensure simultaneousdisplay of the small-angle XRD, wide-angle XRD and DSC results during theircollection (Fig. 4A).

466 C. Lopez et al.

Calorimeter Cell

X-RaysSample

Smal

l-an

gle

dete

ctor

Wide-angle detector

(A)

(B)

T C CT C C

CountingCounting ElectElect..nVmeternVmeterT CtrlT Ctrl

LD2

LD2

Line

arLi

near

dete

ctor

1de

tect

or 1

SampleSampleRefRef..

XX--raysrays(synchrotron)(synchrotron)

MCD

SAXD

WAXD

1770 mm

2))Calorimeter

T C CT C C

CountingCounting ElectElect..nVmeternVmeterT CtrlT Ctrl

LD2

LD2

Line

arLi

near

dete

ctor

1de

tect

or 1

SampleSampleRefRef

XX--raysraysSmall Angles

(synchrotron)(synchrotron)

q = 0 – 0.45 Å-1

MCD

SAXD

WAXD

300 mm

2θ))

Wide Anglesq = 1.1 – 2.1 Å-1

Figure 4. Experimental set-up of the microcalorimeter in the time-resolved synchrotron X-raydiffraction environment. (A) Schematic representation: the cell is positioned with the capillary con-taining the sample perpendicular to the beam in such a way that the diffraction patterns are recordedin the vertical plane by one or two one-dimensional proportional detectors (LD) at small and wideangles. Counting electronic (Counting Elect.), nanovoltmeter (nVmeter) and temperature controller(T Ctrl) are monitored by a single computer. The temperature-controlled cryostat (TCC) is kept atconstant temperature (e.g. 6 ◦C). (B) Set-up on the D22 bench of the synchrotron (LURE, Orsay,France).

XRD patterns were recorded by trans-mission through Lindeman glass capillar-ies (GLAS, Muller, Berlin, Germany), with0.01 mm of wall thickness and diame-ter Φ = 1.40 ± 0.10 mm. These capil-laries were specially designed for X-raystudies since they allow minimum atten-

uation of the beam and parasitic scatter-ing. Furthermore, they are characterised bypoor thermal conductivity and thus guaran-tee minimum thermal losses. Samples wereprepared by loading the capillaries withabout 20 μL of cream or melted AMF us-ing a syringe and a thin Teflon capillary.


The samples in the capillary wereheated to 60 ◦C for 5 min to ensure thatall crystals and nuclei were melted. Then,the samples were cooled with cooling ratesin the range 0.15 ◦C·min−1 � Rcooling �1000 ◦C·min−1. The most rapid Rcooling

were obtained by rapid introduction of thecapillary into the calorimeter pre-cooled tothe temperature, e.g. −8 ◦C or 4 ◦C. Tem-pering in isothermal conditions was alsoperformed, e.g. at −8 ◦C, 4 ◦C and 20 ◦C.Then, XRD patterns were recorded as afunction of time and upon subsequent cool-ing or heating (in general at 2 ◦C·min−1).The samples of concentrated fat globuleswere not cooled at T < −8 ◦C to avoid iceformation.

Each XRD pattern recorded as a func-tion of time (crystallisation and meltingexperiments) was analysed using Peak-fit software (Jandel scientific, Erkrath,Germany). XRD peaks were fitted by theGaussian-Lorentzian (sum) equation, todetermine the position of the maximum,maximal intensity and area under eachXRD peak [11].

2.2.2. DSC experiments

Thermal analyses were conducted byDSC, using a DSC-7 calorimeter (PerkinElmer, St Quentin en Yvelines, France)equipped with an Intracooler II and run-ning under Pyris software and a TA Q 1000calorimeter (TA Instruments, New Castle,DE). About 5 to 10 mg of cream wereweighed in a hermetic aluminium pan of50 μL. An empty pan was used as refer-ence.

2.2.3. Fat globule size measurements

The fat globule size distributions weremeasured by laser light scattering usinga Mastersizer 2000 (Malvern Instruments,

Malvern, UK). From the size distribu-tion, the average volume-weighted diam-eter, d43 = Σnid4

i /Σnid3i (where ni is the

number of fat globules in a size class ofdiameter di), was calculated by the instru-ment software.

3. RESULTS AND DISCUSSION

The organisation of TG molecules inthe solid state (e.g. in fat crystals) wasinvestigated in milk fat globules, by vary-ing the cooling rates. The crystallisa-tion properties of TG in milk fat glob-ules were studied upon slow cooling(0.15 ◦C·min−1), at intermediate coolingrates (0.5 < Rcooling ≤ 3 ◦C·min−1) andafter quenching (∼1000 ◦C·min−1), to char-acterise the most unstable crystalline struc-tures and their reorganisation as a functionof time.

The examination of TG polymorphismin milk fat globules is especially difficultsince (i) both small- and wide-angle XRDshould be considered at the same time andcompared to determine the evolution ofeach of the species as a function of time;(ii) the X-rays diffracted by each of thecrystalline structures are reduced to theproportion of the fraction considered; (iii)the whole XRD signal is largely absorbedby the surrounding water and its solutes(e.g. casein micelles); and (iv) the peakbroadening results from the crystallisationconstraints in dispersed systems.

3.1. Crystallisation in milk fatglobules during slow cooling:Rcooling < 0.5 ◦C·min−1

The crystals formed in milk fat glob-ules upon slow cooling (0.15 ◦C·min−1)and their melting behaviour (2 ◦C·min−1)were considered. For the first time to theauthors’ knowledge, the XRD experimentsperformed at small angles showed that

468 C. Lopez et al.

Figure 5. Structural evolution, expressed in scattering vector q (Å−1), of triacylglycerols dispersedin milk fat globules during cooling from 60 ◦C to −8 ◦C at 0.15 ◦C·min−1. Three-dimensional plotsof the XRD patterns recorded (A) at small angles and (B) at wide angles. (C) Differential scanningcalorimetry curve recorded simultaneously.

crystallisation in fat globules occurs suc-cessively, producing four types of crys-tals [11]. The lamellae formed upon slowcooling correspond to two 2L structures(46.5 and 40 Å) and to two 3L structures(71.3 and 65 Å) (Fig. 5A). Nucleation oc-curs in the α form, then the α + β′ poly-morphic forms coexist until the end of thecooling (Fig. 5B). The DSC crystallisa-tion curve recorded simultaneously showsa single exothermic peak (Fig. 5C). The4 types of crystals start to form within a10 ◦C range, from about 22 ◦C, prevent-ing separation of overlapped peaks by DSCrecording. Before the formation of crys-tals in milk fat globules, characterised bydiffraction peaks, both small- and wide-angle XRD patterns show scattering peaks,respectively, centred at 0.28 Å−1 (22.44 Å)and 1.38 Å−1 (4.55 Å), corresponding to

the X-ray signature of TG in their liquidstate, as described by Larsson [7].

A quantitative analysis of the XRDdata was performed to determine the rel-ative proportion of the 5 different phases,4 solids and 1 liquid, formed in milk fatglobules upon slow cooling and their evo-lution as a function of temperature. It isgenerally accepted that the sum of integralsof the whole diffraction peaks of a phaseis roughly proportional to its abundance ina material [5]. The total diffracted inten-sity is not affected by the effect of crystalperfection, crystal size or the polymorphicform, since X-ray scattering is only propor-tional to the number of atoms present in theorganised system. Thus, we determined theareas for each XRD peak recorded at smallangles during cooling, using Peakfit soft-ware (Fig. 6 left). The relative abundance


of each of the phases was obtained bysumming the areas of each of the small-angle XRD peaks corresponding to thesame crystalline structure (e.g. the 5 areasof peaks corresponding to the 5 orders ofthe 3L1 structure). Figure 6 right showsthe evolution of the relative proportionsof the 5 phases as a function of tempera-ture during cooling of milk fat globules at0.15 ◦C·min−1. The successive occurrencesand growths of the 4 different solid phasesat the expense of the liquid or of one of thephases, newly formed but metastable, areclearly evidenced.

After cooling, the samples were heatedfrom −8 ◦C to 60 ◦C at 2 ◦C·min−1 tostudy the melting behaviour of TG in milkfat globules (Fig. 7). Figure 7A shows theXRD patterns recorded at small and wide(insert) angles during heating. The deter-mination of the evolution of maximal in-tensity of each small-angle XRD peak as afunction of temperature (Fig. 6B) permitsone to relate the structural and the ther-mal events (Fig. 6C). The decrease in in-tensity of the XRD peaks recorded at smallangles corresponds to the melting of thecorresponding crystalline structures. Fromabout 12 ◦C, a new 2L β’ (40 Å) structureformed during the heating, correspondingto the increase in intensity (Fig. 7B) of thepeak observed at small angles (Fig. 7A). Atwide angles, the XRD patterns show α →β’ → liquid transitions (Fig. 7A). This re-crystallisation of TG during heating with apolymorphic transition of monotropic type(α → β’) showed that fat crystals formedduring slow cooling of milk fat globuleswere still metastable. The DSC meltingcurve showed 3 endotherms that corre-spond to the melting of the crystallinestructures, as indicated in Figure 7C. TGdispersed in milk fat globules were in theirliquid state for T > 37 ◦C.

The crystallisation behaviour of AMF insimilar conditions was completely differ-ent [13], showing the influence of the dis-persion state.

3.2. Crystallisation in milk fatglobules for cooling rates0.5 � Rcooling � 3 ◦C·min−1

At intermediate cooling rates, the firstcrystalline structures formed in milk fatglobules are 2L structures (2L1 = 47 Åand 2L2 = 42 Å). Then, the succes-sive formation of 5 sharp XRD peakscorresponds to the crystallisation of TGmolecules in a 3L (71.2 Å) structure (re-sults not shown in this paper; see [13]). The3 lamellar structures that are successivelyformed upon cooling have a hexagonalpacking of the fatty acid chains (α form).At −8 ◦C, the reversible formation of apseudo-orthorhombic perpendicular pack-ing of the acylglycerol chains, called sub-α, was observed. This α → sub-α transi-tion that occurs at low temperature has alsobeen observed in AMF but is favoured inthe dispersed state [16].

The melting behaviour was studiedupon heating at 2 ◦C·min−1. Figure 8Ashows the 3D plot of the small- and wide-(insert) angle XRD patterns. Numerousstructural rearrangements were observedbefore final melting of TG molecules inmilk fat globules, showing that cooling atintermediate rates leads to the formationof unstable crystalline varieties. Roughly,the melting of the α 3L (71.2 Å) struc-ture is first observed and some TG trans-form into a new 3L (65 Å) structure thatmelts rapidly, then the melting of the α 2L(47 and 42 Å) structures is observed. Inthe 16 ◦C � T � 24 ◦C domain, XRDpatterns show a transition and the forma-tion of a new diffraction line, correspond-ing to a β’ 2L (39 Å) structure, before fi-nal melting of TG for T > 40 ◦C. This β’2L (39 Å) structure that crystallises duringheating is probably formed by the reorgan-isation of the TG initially incorporated inthe other lamellar structures formed duringthe cooling process. At wide angles, we ob-served the following transitions upon heat-ing: sub-α ↔ α → β′ → liquid (Fig. 8A,

470 C. Lopez et al.

Liqu

ide

3L1

(005

)

3L2

(002

)

2L2

2L1

3L2

3L1

(003

)

3L1

(002

)

3L1

-8°C

Fig

ure

6.A

naly

sis

ofX

-ray

diff

ract

ion

(XR

D)

patte

rnal

low

ing

the

dete

rmin

atio

nof

the

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tive

prop

ortio

nof

the

crys

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ures

asa

func

tion

ofte

mpe

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re.L

eft:

exam

ple

ofP

eakfi

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lysi

spe

rfor

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smal

l-an

gle

XR

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tter

nre

cord

edat−8

◦ Cat

the

end

ofco

olin

gof

mil

kfa

tglo

bule

sfr

om60◦ C

to−8

◦ Cat

0.15◦ C·m

in−1

(Fig

.5)

.T

hear

eaun

der

the

peak

sco

rres

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toth

esa

me

lam

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ight

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erns

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idon

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eval

uate

dfr

ompe

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at.


Fig

ure

7.M

eltin

gbe

havi

our

oftr

iacy

lgly

cero

lsin

milk

fat

glob

ules

upon

heat

ing

at2◦ C·m

in−1

afte

rsl

owco

olin

gat

0.15

◦ C·m

in−1

.(A

)T

hree

-di

men

sion

alpl

ots

obta

ined

from

the

X-r

aydiff

ract

ion

patt

erns

reco

rded

atsm

alla

ndw

ide

(ins

ert)

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ncti

onof

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ture

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volu

tion

ofth

ein

tens

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sof

the

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l-an

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X-r

aydiff

ract

ion

peak

s,re

cord

eddu

ring

the

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ing

(the

inte

nsit

ies

have

been

expr

esse

das

ape

rcen

tage

ofth

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um).

(C)

Mel

ting

curv

ere

cord

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mul

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eren

tial

scan

ning

calo

rim

etry

.

472 C. Lopez et al.

insert). The evolution of maximal intensi-ties of each XRD peak recorded at bothsmall and wide angles during heating ofcream allowed us to delimit the domainsof existence of the crystals and to relatethe thermal events recorded by DSC to thestructural information recorded by XRD(Fig. 8B) [14].

3.3. Crystals formed afterquenching:Rcooling ∼ 1000 ◦C·min−1

The most unstable crystalline structuresformed by TG molecules in natural milk fatglobules and in AMF were investigated af-ter quenching from 60 ◦C to −8 ◦C [10,12].We identified the formation of 2 lamellarstructures from the liquid: 3L (70.4 Å) anda transient 2L (47 Å) with a lateral organ-isation of the fatty acid chains correspond-ing to the most unstable polymorph, i.e. theα form (hexagonal subcell) (Fig. 9). Theacquisition of XRD patterns as a functionof time after quenching permitted the ob-servation that the α 2L (47 Å) structure wasvery unstable since it disappeared during a20-min isothermal recording.

The crystallographic properties of milkfat globules were also investigated afterquenching from 60 ◦C to 4 ◦C, which isthe temperature of storage of dairy prod-ucts, and followed as a function of time inisothermal conditions at 4 ◦C [15]. Aftersuch a rapid cooling, the main crystallinestructure formed corresponded to an α 3L(70.5 Å). As for quenching at lower tem-peratures (e.g. −8 ◦C), the α 2L (47 Å) wasalso characterised, but it disappeared morerapidly after quenching at 4 ◦C in compar-ison with −8 ◦C.

The rapid cooling of milk fat from60 ◦C to low temperatures, e.g. −8 ◦C or4 ◦C, induces a brusque liquid → solidphase transition. The amount of the liq-uid phase at the temperature of quenchingcan favour the structural rearrangement ofTG molecules. Phase transitions occurred

as a function of time, in isothermal condi-tions, with changes in both the polymor-phic forms and the longitudinal organisa-tion of TG molecules. These polymorphictransitions, e.g. α → β′ → β, were relatedto successive increases in the density of fatglobules during storage at 4 ◦C [15].

Furthermore, we observed that thephase transitions were delayed in fat glob-ules in comparison with AMF [15].

3.4. Crystals formed after temperingin isothermal conditions

The crystals formed in milk fat globulesafter tempering at different temperaturescorresponding to the storage and consump-tion of dairy products were characterised.

The structures formed after storage fort > 5 days at 4 ◦C were identified (Fig. 10).They correspond to 2 lamellar organisa-tions: mainly a 2L (40.5 Å) and a 3L(54.2 Å) structure (Fig. 10A). The lat-eral packing of the fatty acid chains corre-sponds to the coexistence of 4 crystallinepackings: one α form, two β′ forms andtraces of the β form (Fig. 10B). Similarstructures were characterised in milk fatglobules and AMF [15]. The absence ofevolution of the thickness of the lamel-lae upon subsequent heating and the ab-sence of recrystallisation were in favourof the polymorphic stability of the crys-tals formed after this tempering period (re-sults not shown in this paper, see [15]). Theendothermic peaks recorded by DSC uponheating were related to the successive melt-ing of the crystalline structures formed dur-ing the tempering period: the 3L structure,then the 2L structure [15].

This work also evidenced the formationof crystals during storage of milk fat glob-ules (creams) for t > 20 h at room temper-ature (e.g. T ∼ 20 ◦C): 2L (40.5–41.1 Å)lamellar structure [8]. The melting of this2L structure corresponds to a broad en-dothermic peak recorded by DSC [18].Upon subsequent slow cooling of the fat


Figure 8. Structural evolution, expressed in scattering vector q (Å−1), of triacylglycerols dispersedin milk fat globules during heating at 2 ◦C·min−1 after cooling at 1 ◦C·min−1. (A) Three-dimensionalplots of the XRD patterns recorded at small and wide (insert) angles. (B) Differential scanningcalorimetry curve recorded simultaneously.

474 C. Lopez et al.

3L001

3L002

3L003 + 2L002

2L001

0.00 0.06 0.13 0.19 0.26 0.32q (Å-1)

0

1000

2000

I (cp

m)

70.4Å47Å

35.8Å

23.7Å

1.10 1.40 1.70q (Å-1)

0

2e+04

3e+04

4.22Å

4.21Å

3L001

3L002

3L003 + 2L002

2L001

0.00 0.06 0.13 0.19 0.26 0.32q (Å-1)

0

1000

2000

I (cp

m)

70.4Å47Å

35.8Å

23.7Å

1.10 1.40 1.70q (Å-1)

0

2e+04

3e+04

4.22Å

0.00 0.06 0.13 0.19 0.26 0.32q (Å-1)

0

1000

2000

I (cp

m)

70.4Å47Å

35.8Å

23.7Å

1.10 1.40 1.70q (Å-1)

0

2e+04

3e+04

4.22Å

4.21Å

Figure 9. Small- and wide- (insert) angle X-ray diffraction patterns recorded at −8 ◦C after quench-ing of cream (thick line) and anhydrous milk fat (thin line) from 60 ◦C.

globules tempered at 20 ◦C, the amountof crystals in this 2L form increases andtwo additional types of 3L structures areformed (3L1 type: 68.5–70.7 Å; 3L2 type:61.2–64.2 Å), the characteristics of whichdepend on the size of the milk fat glob-ules [20].

The crystalline structures formed after atempering period, mainly the 2L (40–41 Å)structure, correspond to stable polymor-phic species and are characterised by ahigher final melting point [18].

3.5. Influence of milk fat globulesize on their crystallisationproperties

The thermal and crystallographic prop-erties of creams were analysed as a func-tion of temperature and time with thewhole droplet size distribution of naturalmilk fat globules [10,11,14,15]. In this partof the work, we focused on the influence offat globule size on TG crystallisation andpolymorphism.

Natural milk fat globules (i.e. coveredby the MFGM) of smaller size (∼1–3 μm)and larger size (∼5–7 μm) were selectedfrom the same initial milk (fat globules∼4 μm) by using a novel microfiltrationprocess [21]. Figure 11A shows that theDSC crystallisation curves recorded forcreams containing natural milk fat glob-ules with different sizes correspond to asingle exothermic peak and that the ini-tial temperature of crystallisation (Tonset)is delayed for smaller fat globules. Fig-ure 11B shows the decrease in Tonset asa function of the decrease in natural milkfat globule size [20]. Lopez et al. [14]prepared emulsions by homogenising milkfat with β-lactoglobulin and using differ-ent pressures to obtain significantly differ-ent average droplet sizes. The latter authorsshowed that reducing the size of the emul-sion droplets induces a higher supercool-ing and that the Tonset is displaced towardlower temperatures. The delay in crystalli-sation observed for natural milk fat glob-ules and emulsion droplets was indepen-dent of the composition of the milk fat and


0.02 0.10 0.17 0.25 0.32 0.40q (Å-1)

1000

2000

3000

4000

I (co

unts

)

54.2 Å

40.5 Å

28.8Å

2L

3L

1.10 1.30 1.50 1.70 1.90 2.10q (Å

3.60

7.20

10.80

14.40

18.00

(Tho

usan

ds)

I (co

unts

) (4.65(4.65ÅÅ))

(4.37(4.37ÅÅ))

(4.25(4.25ÅÅ))(4.15(4.15ÅÅ))

(3.83(3.83ÅÅ))(3.783.78ÅÅ)

ββ

αα

ββ ββ' '22

'

11'22

ββ

ββ 11

-1)

(B) Longitudinal organisation

(A) Lateral packing

0.02 0.10 0.17 0.25 0.32 0.40q (Å-1)

1000

2000

3000

4000

I (co

unts

)

54.2 Å

40.5 Å

28.8Å

2L

3L

1.10 1.30 1.50 1.70 1.90 2.10q (Å

3.60

7.20

10.80

14.40

18.00

(Tho

usan

ds)

I (co

unts

) (4.65(4.65ÅÅ))

(4.37(4.37ÅÅ))

(4.25(4.25ÅÅ))(4.15(4.15ÅÅ))

(3.83(3.83ÅÅ))(3.783.78ÅÅ)

ββ

αα

ββ ββ' '22

'

11'22

ββ

ββ 11

-1)


(A) Lateral packing

0.02 0.10 0.17 0.25 0.32 0.40q (Å-1)

1000

2000

3000

4000

I (co

unts

)

54.2 Å

40.5 Å

28.8Å

2L

3L

1.10 1.30 1.50 1.70 1.90 2.10q (Å

3.60

7.20

10.80

14.40

18.00

(Tho

usan

ds)

I (co

unts

) (4.65(4.65ÅÅ))

(4.37(4.37ÅÅ))

(4.25(4.25ÅÅ))(4.15(4.15ÅÅ))

(3.83(3.83ÅÅ))(3.783.78ÅÅ)

ββ

αα

ββ ββ' '22

'

11'22

ββ

ββ 11

-1)-1)


(A) Lateral packing

Figure 10. X-ray diffraction patterns recorded at wide angles (A) and small angles (B) after storageof cream (thick line) and anhydrous milk fat (thin line) for 5 days at 4 ◦C.

476 C. Lopez et al.

(A) DSC crystallisation curves

(B) Initial temperature of crystallisation

1515

1616

1717

1818

1919

2020

2121

2222

00 11 22 33 44 55 66 77 88

d43 (µm)

T ons

et(°

C)

--1010 00 1010 2020 3030 4040Temperature (°C)

0.98

1.00

1.02

1.04

1.06

1.08

1.10

HF

(a.u

.) E

ndo

> (d43

= 1.75 µm)

(d43 = 5.41 µm)

(d43 = 0.93 µm)

1°C/min

(A) DSC crystallisation curves

(B) Initial temperature of crystallisation

1515

1616

1717

1818

1919

2020

2121

2222

00 11 22 33 44 55 66 77 88

d43 (µm)

T ons

et(°

C)

--1010 00 1010 2020 3030 4040Temperature (°C)

0.98

1.00

1.02

1.04

1.06

1.08

1.10

HF

(a.u

.) E

ndo

> (d43

= 1.75 µm)

(d43 = 5.41 µm)

(d43 = 0.93 µm)

1°C/min

Figure 11. Crystallisation properties of natural milk fat globules as a function of their size.(A) Differential scanning calorimetry (DSC) curves recorded upon cooling of milk fat globulesat 1 ◦C·min−1. The size of small fat globules is indicated in the figure. (B) Evolution of the initialtemperature of crystallisation as a function of natural milk fat globule diameter (d43) calculatedfrom the DSC crystallisation curves.


the surface of droplets (MFGM vs. milkproteins) and was related to the size ofdispersed fat particles. This delay in crys-tallisation can be explained by the the-ory of heterogeneous nucleation in dis-persed systems, e.g. initiated by catalyticimpurities. In emulsions such as creams, atleast one nucleus must be formed in eachdroplet to achieve full crystallisation, andthe time needed to obtain the nucleus isinversely proportional to globule volume.Consequently, a lower temperature due toa higher supercooling is needed to inducecrystallisation in a dispersed system withsmaller fat globules since more catalyticimpurities are required [29].

Lopez et al. [14] showed that what-ever the size of fat globules, crystallisationmainly occurs in the α 3L (72 Å) uponcooling at 1 ◦C·min−1. However, the de-crease in the fat globule size induces (i) theformation of less 2L structures and (ii) adecrease in small-angle XRD peak maxi-mum intensity correlated with an increasein peak width. This was interpreted as re-sulting from defaults in the organisationof TG molecules in the crystals, either di-rectly due to the curvature of the surfacefrom which crystals are supposed to grow,or indirectly to the faster relaxation thatcan also induce the formation of crystalsof smaller size.

This work showed that there are no ma-jor differences in the type of crystallinestructures formed by TG, after eliminatingthermal history. However, the level of or-ganisation of TG in the crystals and/or thesize of the crystals can differ as a functionof the size of the fat globules. As a sum-mary, the smaller the fat globule size, thelarger the supercooling, the faster the crys-tals’ growth and the larger the crystal dis-organisation inside the globules.

Differences in the crystallisation prop-erties were observed after tempering milkfat globules of different sizes at 20 ◦Cand subsequent cooling at 0.5 ◦C·min−1 to−8 ◦C (Fig. 12). The smaller fat globules

contained much more 3L structures andless 2L structures than larger ones [20].Moreover, the crystals formed in the largerfat globules (d43 = 7.15 μm) melted uponheating but did not evolve from a polymor-phic point of view, whereas the crystalsformed in the smaller fat globules (d43 =0.93 μm) were metastable and reorgan-ised to form more stable species before fi-nal melting of TG. These differences couldhave interesting applications in the manu-facture of dairy products in which temper-ing periods are involved in the technologi-cal process (e.g. butter, whipped cream, icecream).

3.6. Compound crystals are formedin milk fat globules

The studies performed on milk fat glob-ule crystallisation showed that it occurssuccessively, producing 1, 2, 3 or 4 dif-ferent varieties depending on the coolingrate and that only small-angle XRD exper-iments permit the characterisation of thesedifferent types of crystals [10, 11, 14, 15].We showed that upon cooling the first lon-gitudinal organisations of TG moleculesdispersed in fat globules correspond to2L lamellar structures with long spacingsof 40–42 and 46–48 Å. These organisa-tions may correspond to crystallisation ofhigh-melting point TG, with saturated andsimilar chain length fatty acids (e.g. PPS,PPP, MPP; P: palmitic acid, S: stearic acid,M: myristic acid). Then, crystallisation of3L structures occurs: 3L1 = 70–72 Å and3L2 = 65 Å. They may correspond tocrystallisation of TG molecules with un-saturated fatty acids, or to TG with fattyacid chains of different lengths (e.g. BuPP,BuPO, PPO; Bu: butyric acid, O: oleicacid). Recently, Lopez et al. [19] showedthat the 2L structures are formed by thestearin fraction, whereas the 3L structuresare formed by the olein fraction, after dry

478 C. Lopez et al.

Large fat globules

Small fat globules

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

q (Å-1)

(Tho

usan

ds)

Inte

nsity

(a.u

.)

3L13L2

2L

3L1 (002)

3L2 (002)

3L1 (005)

3L2 (005)

3L13L2

2L

3L1 (002)

3L2 (002)

3L1 (003)

3L1 (005)

0

9

18

27

36

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100

Small fat globules d43=0.93 µm

Large fat globules d43=7.15 µm

Vol

ume

(%)

Diameter (µm)

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 1000

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100



Vol

ume

(%)

Diameter (µm)

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

q (Å-1)

(Tho

usan

ds)

Inte

nsity

(a.u

.)

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

q (Å-1)

()

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60

q (Å-1)

()

3L13L2

2L

3L1 (002)

3L2 (002)

3L1 (005)

3L2 (005)

3L13L2

2L

3L1 (002)

3L2 (002)

3L1 (003)

3L1 (005)

0

9

18

27

36

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100



Vol

ume

(%)

Diameter (µm)

0

2

4

6

8

10

12

14

16

0,01 0,1 1 10 1000

2

4

6

8

10

12

14

16

0,01 0,1 1 10 100

d43=0.93 µm

d43=7.15 µm

Vol

ume

(%)

(µm)

Figure 12. Comparison of the small-angle X-ray diffraction patterns recorded at −8 ◦C with smalland large natural milk fat globules that were initially stabilised at 20 ◦C for 24 h and subsequentlycooled at 0.5 ◦C·min−1 down to −8 ◦C. Insert shows the size distribution of milk fat globules.

fractionation from the same initial batch ofmilk fat, performed at 21 ◦C.

The comparison of the small numberof crystal types formed (4 longitudinalorganisations and 5 polymorphic formsidentified) with the large number of TGmolecules present in milk fat (200 TGspecies were identified) provides evidencethat each of the varieties corresponds infact to compound crystals. The forma-tion of compound crystals, also calledmixed crystals as they contain different TGmolecular species in the crystal lattice, isnot surprising in a natural fat with a widecompositional range of TG molecules,such as milk fat.

4. CONCLUSION

The use of high-flux X-ray sources (syn-chrotron) permitted the study of the poly-morphism and phase transitions displayedas a function of temperature and time bythe complex mixture of TG dispersed inmilk fat globules. XRD experiments per-formed at both small and wide anglesallowed the identification on a molecu-lar scale of the TG longitudinal stack-ing and the lateral packing of fatty acidchains in lamellar structures, respectively.Thanks to the resolution obtained with thevarious set-ups used in these studies, thesmall-angle XRD analysis performed forthe first time on milk fat in the dispersed


state was essential for the identificationof the longitudinal organisation of TGmolecules in the lamellar structures andtheir time- and temperature-induced tran-sitions. Earlier studies concerning milk fatpolymorphism were based on the descrip-tion of XRD peaks corresponding to thelateral packing of TG, and permitted onlythe identification of the crystalline sub-cells. Furthermore, we showed that differ-ent types of crystals (1 to 4) and severalpolymorphic forms (1 to 5) coexist in milkfat globules at low temperatures, togetherwith a liquid phase. The coupling of XRDwith DSC recordings permitted us to re-late the structural transitions to the ther-mal behaviour of milk fat globules. Thesetools allowed, for the first time to ourknowledge, the identification and the char-acterisation of crystalline structures withinwater-dispersed and highly complex, natu-ral or processed food products as well astheir transitions (several studies on cheeseand ice cream are under way). Moreover,the comparison of the crystallisation prop-erties in the dispersed state and in bulk pro-vides information on the influence of theinterface on crystallisation and its compo-sition (Lopez et al., in preparation). Thiswork was performed on bovine milk fatglobules but similar work is under way forother species: dromedary [17], goat [2],ewe and buffalo.

Acknowledgements: The authors gratefullyacknowledge the French dairy board ARILAITRecherches (Paris, France) for supporting thisresearch and all the members of the steeringcommittee on fat research. They also thankM.C. Michalski, N. Leconte and V. Briard(UMR STLO, INRA, Rennes, France) for col-laborating in the studies on the crystallisationproperties of natural milk fat globules as a func-tion of their size.

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