Milk fat and primary fractions obtained by dry fractionation

Chemistry and Physics of Lipids 144 (2006) 17–33

Milk fat and primary fractions obtained by dry fractionation 1. Chemical composition and crystallisation properties Christelle Lopez a,b,∗ , Claudie Bourgaux c , Pierre Lesieur c , Alain Riaublanc d , Michel Ollivon a a

Equipe Physico-chimie des Syst`emes Polyphas´es, UMR 8612 du CNRS, 5 rue J.B. Cl´ement, 92296 Chˆatenay-Malabry, France b Equipe Physico-chimie & Biochimie, UMR 1253 STLO INRA Agrocampus, 65 rue de Saint-Brieuc, 35042 Rennes, France c Laboratoire pour l’Utilisation du Rayonnement Electromagn´ etique, Bat. 209D Universit´e Paris-sud, 91898 Orsay, France d Laboratoire d’Etude des Interactions des Mol´ ecules Alimentaires, INRA, Rue de la G´eraudi`ere, 44315 Nantes, France Received 10 February 2006; received in revised form 26 May 2006; accepted 8 June 2006 Available online 27 June 2006

Abstract The chemical composition and crystallisation properties of milk fat and its primary fractions, obtained by dry fractionation at 21 ◦ C, were investigated. The solid fraction (stearin) and the liquid fraction (olein) displayed a different triacylglycerol (TG) composition. Stearin fraction was enriched in long-chain fatty acids, whereas olein fraction was enriched in short-chain and unsaturated fatty acids. Crystallisation properties of milk fat, and both the stearin and olein fractions were studied on cooling at |dT/dt| = 1 ◦ C min−1 by differential scanning calorimetry and time-resolved synchrotron X-ray diffraction (XRD) at small and wide angles. Two main ˚ and ␤ types of crystals corresponding to double chain length structures were characterised in the stearin fraction: ␣ 2L1 (47.5 A) ˚ ˚ 2L2 (41.7 A). A triple chain length structure was formed in the olein fraction: ␣ 3L (72.1 A). Crystallisaton of milk fat showed the ˚ and one 3L (72.1 A) ˚ lamellar structures with an hexagonal packing (␣ form). A schematic formation of two 2L (47.3 and 41.6 A) representation of the 3L packing of olein fraction was proposed to explain how a wide diversity of TG can accommodate to form a ˚ Furthermore, the sharpness of the small-angle XRD lines associated to the ␣ form was lamellar structure with a thickness of 72 A. explained by the formation of liquid crystals of smectic type. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Triglyceride; Dry fractionation; X-ray diffraction; Differential scanning calorimetry; Crystals

1. Introduction Milk fat is composed of 98% triacylglycerols (TG), which are triesters of fatty acids and glycerol. More than 400 fatty acids and 200 TG have been identified (Gresti et al., 1993). Undoubtedly, it is one of the most com-

∗ Corresponding author. Tel.: +33 2 23 48 56 17; fax: +33 2 23 48 53 50. E-mail address: [email protected] (C. Lopez).

plex fats found in nature. Milk fat is characterised by short-chain (C4–C8: 8.3%), medium-chain (C10–C12: 6.6%) and long-chain (C14–C18: 81.9%) length fatty acids (Jensen and Newburg, 1995). Moreover, milk fat is a relatively high saturated fat: about 65% saturated fatty acids (mainly C16:0, C18:0 and C14:0) and about 35% unsaturated fatty acids (mainly C18:1). The chemical composition of milk fat varies greatly with the season, breed, stage of lactation and the type of feed (Walstra and Jenness, 1984). Naturally occurring seasonal variations can have a major effect on the physical stability of milk

0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2006.06.002

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fat globules, on the functional properties of fat and on the final texture of fat-rich products (Walstra and Jenness, 1984). Thus, the control of fat-rich products quality, e.g. butter, in different seasons and from different origins of cream, is a real challenge for the industrials. Regarding its sensorial, textural and functional properties, milk fat is important in dairy products, i.e. cream, cheese, butter and ice-cream. However, milk fat has several attributes which limit its consumption and its use. Among these are the negative health aspects due to its high concentration of saturated fatty acids and cholesterol, its high price and its limited functionality as compared to other tailored fats (O’Donnell, 1993). Modification of milk fat composition to improve its nutritional and functional properties is of considerable interest for expanding its use in the food industry. In this respect, numerous techniques have been applied including dietary, chemical and physical. Dietary manipulation by means of feeding dairy animals can produce milk fat with a high content of polyunsaturated fatty acids (Palmquist et al., 1993; Chilliard et al., 2001). However, it has not been commercially successful due to the high cost of the supplement. Furthermore, the resultant milk fat may be more susceptible to oxidative rancidity which may lead to sensory defaults. Modifying milk fat by chemical methods such as hydrogenation or enzymatic interesterification has been reported (Rousseau et al., 1996; Pal et al., 2000). Although these processes have been permitted by food regulations, they are not popular because of the consumer demand for edible fats devoid of any chemical treatments and with natural characteristics. Physical modification of milk fat can be divided into (i) processes that alter the chemical composition of the fat, as fractionation techniques and blending with vegetable oils (Ali and Dimick, 1994; Simoneau et al., 1994) and (ii) processes that do not alter the chemical composition of the fat, as texturisation and cream tempering treatments (Kleyn, 1992). Milk fat can be fractionated by molecular distillation (Arul et al., 1988; Campos et al., 2003), supercritical carbon dioxide extraction (Chen et al., 1992; Bhaskar et al., 1993) and by crystallisation from the melted fat with and without solvents and detergents (Marangoni and Lencki, 1998; van Aken et al., 1999). However, fractionation by short-path distillation and by supercritical carbon dioxide are expensive (Boudreau and Arul, 1993). The use of solvents or surfactants is more efficient in the separation between the solid and liquid fractions by reducing the entrainment. Nevertheless, it is not preferred in food applications owing to the high cost of solvent removal and disposal, flavour and toxicological problems that may arise from solvent residues and the loss of natural flavour com-

pounds when deodorizing the fractions. Moreover, the use of organic solvents is not permitted in the dairy industry. Considering the limitation imposed on the fractionation techniques, one potential approach, which has been commonly employed and exploited commercially by the dairy industry, is melt crystallisation. Some of its benefits include the absence of organic solvents or process aids (dry fractionation), the reasonable cost of processing, a relatively simple equipment and preservation of the delicate flavour of milk fat which confirms with the “natural” product image (Fatouh et al., 2003). Dry fractionation involves two main steps, crystallisation and separation. Techniques using diverse materials as membranes have been employed in the absence of solvent to separate crystals from the liquid matrix (Kaylegian and Lindsay, 1995). When milk fat TG are crystallised at temperatures below their melting points, the resultant slurry is filtered by vacuum filtration (Amer et al., 1985), pressure filtration (Patience et al., 1999) or filter centrifugation (Breeding and Marshall, 1995). The process parameters including cooling rate, agitation rate and final temperature greatly influence the chemical composition and physical attributes of the fractions obtained (Grall and Hartel, 1992). Fractionation of milk fat permits to obtain fractions with variable TG compositions and melting points. Thus, fractionation of milk fat and recombination of the fractions allow the control and the improvement of the thermal and physical properties, i.e. its consistency, and the development of new products. The high melting fractions have found extensive use as shortening in puff pastry imparting a desirable butter flavour, fat bloom inhibitors in chocolate, hard stock for ghee production, cocoa butter replacement in confectionary products, edible films and frozen desserts (Crabtree, 1991; Bystrom and Hartel, 1994; Versteeg et al., 1994; Full et al., 1996; Abd ElRahman et al., 1997). Other potential applications for low melting fractions include biscuits, short breads, cold spreadable butter, pourable frying oils and improving the reconstitutibility of milk powder (Kaylegian and Lindsay, 1995). Many authors have studied the thermal behaviour of milk fat using differential scanning calorimetry (DSC). They showed that it crystallises and melts in several steps that correspond to separate group of TG (Timms, 1980; Marangoni and Lencki, 1998). The complex DSC recordings result from (i) the broad distribution of TG composition, (ii) compound crystallisation meaning that the composition of the crystals is changing during melting and (iii) the existence of a polymorphism of monotropic type for each TG group constituting the

C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

crystal (Walstra and van Beresteyn, 1975; Small, 1986). The consequence of this complexity is that milk fat has a broad crystallisation and melting range (about −40 to 40 ◦ C) and no true melting point. X-ray diffraction (XRD) is an essential tool for elucidating polymorphism of fats since it complements DSC. Polymorphism results from the different possibilities of lateral packing of the fatty acid chains and of longitudinal stacking of molecules in lamellae (Sato, 1996). The longitudinal stacking of the TG molecules and the lateral packing of their acylglycerol chains are recorded by XRD at small and wide angles, respectively, as detailed in Lopez et al. (2000). Recently, anhydrous milk fat crystallisation was monitored by coupled XRD as a function of temperature and DSC techniques (Lavigne, 1995; Lopez et al., 2001a,b, 2002, 2005). Although many studies have focussed on the milk fat fractionation process and on the chemical and thermal characteristics of the separated fractions to improve the understanding of their characteristics, so far only a few studies have been reported about the crystallographic characteristics of the milk fat fractions (Lavigne, 1995). Characterising phase behaviour and polymorphism of fractions and their mixture in milk fat will contribute to a better prediction and control of the physical and rheological properties of food products. The objectives of this study were to characterise the chemical composition of stearin fraction and olein fraction obtained by dry fractionation from the same batch of anhydrous milk fat, and to identify the crystalline structures formed by TG molecules on cooling at |dT/dt| = 1 ◦ C min−1 . This paper is the first of a series examining the thermal and structural properties of milk fat and its primary fractions. 2. Experimental procedures 2.1. Milk fat, stearin fraction and olein fraction Anhydrous milk fat and its primary fractions, stearin and olein, were provided by Fl´echard S.A. (La Chapelle d’Andaine, France). Anhydrous milk fat was industrially fractionated at 21 ◦ C by first slowly cooling melted milk fat during one day in large batch (about 20 t) under very slow agitation (about 3 rpm). This slow crystallisation allows the formation of large spherulites that favour easy filtration (Marangoni, 2005). Second using the dry-fractionation process developed by Tirtiaux (Belgium) which consists in sucking olein oil under low vacuum (500–700 mbar) from crystallised milk fat cake deposited on a permeable thin stainless steel metallic belt. From the same batch of milk fat, the solid fraction

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corresponded to the stearin, whereas the liquid fraction was the olein. 2.2. Chemical composition Determination of the different lipid fractions contained in milk fat and its primary fractions was performed by gas chromatography (GC) after trimethylsilyl derivatisation. About 100 ␮g of lipids were derivatised with 100 ␮l of Tri-Sil/BSA reagent (Pierce, Rockford, USA) during 30 min at ambient temperature. Lipid fractions were then separated using a 12 m DB5 HT column (Ø = 0.32 mm and e = 0.1 ␮m; JW Scientific, Folsom, USA) with a decreasing speed temperature gradient from 100 to 335 ◦ C with a Hewlett-Packard HP5890 Gas chromatograph. Peaks of different fatty acids, monoacylglycerols, diacylglycerols, triacylglycerols, free and esterified cholesterol were quantified using a FID detector, adjusted to take into account differences in response factor and clustered by lipid class. Phospholipid concentration in total lipid samples was determined by phosphorus quantification according to Bartlett (1959). The TG composition of milk fat and both stearin and olein fractions were obtained using reversedphase high-performance liquid chromatography (HPLC) (Gilson Inc., Middleton, USA) with two end capped C18 columns connected in series (Lichrospher 100, 250 mm × 4 mm; Merck, Darmstadt, Germany). Twenty-five microliters of a 10% lipid solution in hexane was injected and then TG species were sequentially eluted by a gradient of chloroform in acetonitrile from 5 to 40% in 190 min then to 50% in 10 min. TG species were detected in effluent by diffraction light scattering (Sedex 55, Sedere, France). Peaks surface (S in ␮V s) were corrected to take into account the non-linearity response of this detector using the following equation: injected quantity (␮g) = 0.0018 × (peak surface)0.6133 . Identification of the species present in each peak was performed in two steps, first major peaks were collected in effluent column and analysed for fatty acid composition as fatty acid methyl esters (FAME) by GC on a DB 225 column (L = 30 m, Ø = 0.32 mm and e = 0.1 ␮m; JW Scientific). Then fatty acids were paired by three according to their relative concentration in the peak. For other species, theoretical distribution of TG was calculated from data on fatty acids distribution on each glycerol position. In HPLC, for each species theoretical molar concentration of which exceed 0.1%, hydrophobic carbon number (HCN) was calculated (HCN = number of carbon in acyl chains − 2 × number of unsaturation). This parameter is linearly related to retention time in RP-HPLC and was validated with identified peaks. So

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C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

retention time for these species were calculated and compared with profiles. On RP-HPLC profiles, we could detect about 120 peaks but only 56 could be identified directly or by comparing with HCN values. So only major species of TG were taken into account in presented table.

determine the maximal intensity as a function of time and the middle-height peak width as a function of diffraction order, as previously described (Lopez et al., 2001c). CaRIne Crystallography 3.0 software (Divergent, Compi`egne, France) was used to calculate a smallangle XRD pattern from the electronic density profile of the 3L lamellar structure proposed.

2.3. XRDT/DSC measurements 2.4. Thermal analysis Experiments were performed using a technique that allows simultaneous synchrotron radiation X-ray diffraction as a function of temperature (XRDT) and high-sensitivity differential scanning calorimetry (DSC) to be carried out in the same apparatus (Keller et al., 1998; Ollivon et al., in press). The experiments were carried out on D22 and D24 benches of synchrotron of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagn´etique, Orsay, France). On D22 bench, two one-dimensional position-sensitive proportional detectors were used for detection simultaneously ˚ −1 ) and wide (q = 1.0–2.2 A ˚ −1 ) at small (q = 0–0.45 A angles. This experimental set-up was already detailed in Lopez et al. (2001a). On D24 bench, a single detector was ˚ −1 ). used for XRD detection at small angles (q = 0–0.55 A In order to make the X-ray data comparison easier, all patterns are presented as a function of the scattering vec˚ −1 , θ (◦ ) the angle tor q (q = 4π sin(θ)/λ; where q is in A of incidence of X-ray relative to the crystalline plane and λ is the X-ray wavelength). DSC thermal measurements and X-ray data were synchronously collected versus time by a single PC microcomputer. The channel to scattering vector q calibration of the detectors was made at wide angles with crystalline β form of high-purity tristearin and at small angles with silver behenate, as previously described (Lopez et al., 2001c). All XRD patterns were recorded by transmission through glass capillaries (GLAS, Muller, Berlin, Germany). Samples were prepared by loading about 20 ␮l of the melted fats in these thin Lindeman glass capillaries with 0.01 mm of wall thickness and diameter Ø = 1.40 ± 0.10 mm using a syringe and a thin Teflon capillary. The samples in the capillaries were heated at 70 ◦ C for 5 min to melt all existing crystals and nuclei; at 70 ◦ C, all the TG are in a liquid state. The crystallisation properties of milk fat, olein fraction and stearin fraction were conducted on cooling from 70 to −7 ◦ C at |dT/dt| = 1 ◦ C min−1 which is an intermediate rate between fast and slow cooling. Peakfit Software (Jandel Scientific, Erkrath, Germany) was used to analyse the XRDT patterns. The XRD peaks recorded at small angles were fitted with the Gaussian–Lorentzien sum (amplitude) equation to

Thermal analyses were conducted with a DSC-7 Perkin-Elmer (St. Quentin en Yvelines, France) using aluminium pans of 50 ␮l (pan, Part No. B014-3021 and cover, Part No. B014-3004) hermetically sealed. An empty, hermetically sealed aluminium pan was used as reference. The calibration of the calorimeter was made with lauric acid (melting point = 43.7 ◦ C, Hmelting = 8.53 kcal mol−1 ). Two protocols were used to study the thermal characteristics of milk fat and its primary fractions. Protocol 1: The samples were heated 5 min at 70 ◦ C then cooled from 70 to −50 ◦ C at |dT/dt| = 1 ◦ C min−1 . Protocol 2: The samples were heated 5 min at 70 ◦ C then cooled from 70 to −7 ◦ C at |dT/dt| = 1 ◦ C min−1 . 3. Results Milk fat fractionation is an industrial process employed to obtain butters or dairy products with specifically adapted thermal properties. This study analyses the relation between fraction compositions of milk fat industrially fractionated at 21 ◦ C and their thermal and structural properties. 3.1. Chemical composition Table 1 shows the chemical composition of milk fat, olein fraction and stearin fraction. They were composed by more than 98% of TG and diacylglycerols. The Table 1 Chemical composition of olein fraction and stearin fraction obtained by dry fractionation from the same batch of milk fat

Triacylglycerol + diacylglycerol Free fatty acid Monoacylglycerol Cholesterol (free and esterified) Phospholipid

Milk fat (%)

Olein fraction (%)

Stearin fraction (%)

98.3

98.5

98.6

0.6 0.4 0.7

0.6 0.4 0.5

0.6 0.2 0.6

<0.04

<0.04

<0.04

C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

other minor components were free fatty acids, monoacylglycerol and cholesterol (the relatively high values of these industrial milk fat and fractions result from butter lipolysis). Table 2 shows the TG composition of milk fat, olein fraction and stearin fraction, which have been determined as a function of the number of carbon by gas chromatography. All the milk fat TG were present in both the liquid and solid fractions but their proportions were different, as a result of dry fractionation performed at 21 ◦ C. The relative TG compositional differences between stearin and olein fractions are shown Fig. 1. The characteristic TG compositional differences were as follows (Table 2; Fig. 1): - Stearin fraction was enriched in TG with: (i) three saturated long-chain fatty acids (SSS, PSS, PPS, PPP, MSS, MPS, MMS, MPP, SSL), (ii) one or two saturated medium-chain fatty acids and a saturated long-

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chain fatty acid (MMS, MMP, LaPS, LaPP, LaMP, CPS, CPP, CMS) and (iii) one monounsaturated longchain fatty acid and two saturated long-chain fatty acids (PoSS, PPoS, LaOS, PSO). Stearin fraction was not enriched in TG with a short-chain fatty acid, except BPPo (1.35% in stearin fraction versus 0.44% in olein fraction) and CaPS (0.83% versus 0.14% in olein). However, stearin fraction contained high levels of TG with a short-chain fatty acid such as butyric acid and capro¨ıc acid: BPO/CaMP (3.26% versus 6.41% in olein), BPP/BMS (3.77% versus 7.33% in olein), CaPP/BOS/CaMS (2.54% versus 3.94% in olein) and BPS (1.81% versus 3.10% in olein) (Table 2). - Olein fraction was enriched in TG with: (i) two monounsaturated long-chain fatty acids (SOO, PoSO, POO, MOO, PPoO) and (ii) one monounsaturated and two medium-chain fatty acids (CMO, CPO, LaMO, MMO, LaPO). However, stearin fraction contained

Table 2 Triacylglycerol composition (mass%) of milk fat and its primary fractions, olein and stearin fractions, obtained by dry-fractionation at 21 ◦ C from main fatty acids (not taking into account the minor fatty acids like ramified, trans and odd numbered ones) Triacylglycerola

Milk fat

Olein fraction

Stearin fraction

Triacylglycerola

BCM BCO BCP BMPo/BLaO BLaP/BMM BPoO BMPd BPPo BMO/CaLaP BMP BOO/CaMO BPO/CaMP BPP/BMS CaPdP CaOO CaPO/CLaO CaPP/BOS/CaMS BPS CyOO CMO CyOP/CMP CyPP CaPS LaOL COO/MMoO CPO/LaMO MMoP/CyOS LaMP/CPP/CMS

0.19 0.20 0.42 0.48 1.15 0.46 0.39 0.63 2.03 3.19 1.67 4.40 5.39 0.40 0.83 2.62 2.93 2.40 1.06 1.32 2.27 1.22 1.18 0.45 0.63 2.38 0.86 2.68

0.26 0.26 0.48 0.76 1.54 0.78 0.97 0.44 2.97 4.63 2.62 6.41 7.33 0.79 1.40 3.78 3.94 3.10 1.01 2.39 1.09 1.13 0.14 0.46 0.59 2.58 0.91 2.26

0.15 0.18 0.24 0.41 0.85 0.22 0.43 1.35 2.62 0.44 1.11 3.26 3.77 0.32 0.59 1.90 2.54 1.81 0.56 0.52 1.80 1.08 0.83 0.31 0.41 1.55 0.54 3.40

CyPS LaPdO/LaPdP/LMO MPL LaOO/MoPO MMO/LaPO COS/MPPo MMP/LaPP/CPS MPdO/MPdP MOO/PPoO MSL/PPL MPO/PPPo LaOS MMS/MPP/LaPS MMP/PdPP/OOO POO PoSO PPO/MSO PPoS PPP/MPS PdOS/PdPS SOO SSL PSO PoSS PPS/MSS SSO PSS SSS

Total a

Milk fat

Olein fraction

Stearin fraction

0.56 0.17 1.26 1.08 3.13 1.35 3.21 1.04 3.08 1.44 5.23 1.47 3.84 1.63 4.14 1.87 5.83 1.45 3.06 0.58 1.24 0.74 3.45 0.89 2.32 0.72 1.14 0.23

0.45 0.48 1.51 1.30 3.63 1.18 1.91 0.90 3.18 1.28 5.10 0.88 1.62 1.44 4.04 1.88 5.38 0.60 0.99 0.37 1.35 0.69 3.18 0.30 0.45 0.65 0.18 0.07

0.51 0.28 0.92 0.70 2.49 1.31 5.19 0.64 2.00 0.61 4.60 1.69 7.14 1.53 2.91 1.25 6.06 2.74 7.04 0.77 0.78 0.80 3.72 1.71 5.47 0.67 2.73 0.53

100.00

100.00

100.00

Abbreviations—B: butyric acid (C4:0); Ca: capro¨ıc acid (C6:0); Cy: caprylic acid (C8:0); C: capric acid (C10:0); La: lauric acid (C12:0); M: myristic acid (C14:0); Pd: pentadecanoic acid (C15:0); P: palmitic acid (C16:0); Po: palmitoleic acid (C16:1); S: stearic acid (C18:0); O: oleic acid (C18:1).

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Fig. 1. Relative triglyceride compositional difference between stearin fraction and olein fraction. Arrows indicate that the triglyceride is present with a concentration ≥2% in one of the two fractions (white arrows), in the two fractions (black arrows).

MOO/PPoO (2.00% versus 3.18% in olein), POO (2.91% versus 4.04% in olein) and MMO/LaPO (2.49% versus 3.63% in olein). Furthermore, olein fraction was enriched in TG characterised by a shortchain fatty acid, butyric or capro¨ıc acid and (i) two sat-

urated long-chain fatty acids (BMP, BPS, BPP, BMS; CaMP, CaPP, CaMS) or (ii) one saturated long-chain fatty acid and one monounsaturated fatty acid (BOS, BPO, BMO; CaMO, CaPO) and (iii) two monounsaturated long-chain fatty acids (BOO).

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TG with one monounsaturated and two saturated long-chain fatty acids were present in both the stearin and olein fractions: PPO/MSO (5.38% in olein versus 6.06% in stearin), PSO (3.18% in olein versus 3.72% in stearin) and MPO/PPPo (5.10% in olein versus 4.60% in stearin). 3.2. Thermal properties on cooling at |dT/dt| = 1 ◦ C min−1 Fig. 2 shows the crystallisation curves recorded using DSC on cooling of milk fat, stearin fraction and olein fraction at |dT/dt| = 1 ◦ C min−1 from 70 to −50 ◦ C. The DSC curves showed different exothermic events and range of crystallisation. The DSC curve recorded upon cooling of milk fat showed at least two wellseparated exotherms. Crystallisation of milk fat started at 20 ◦ C with a sharp exotherm which spaned between 20 and 16.5 ◦ C. The second exotherm recorded on cooling spaned from 14 to about −45 ◦ C. Crystallisation of the olein fraction started at 11.5 ◦ C with a sharp exothermic event formed by two overlapped peaks which spaned to about 2 ◦ C. From 2 to about −30 ◦ C, an exothermic signal was recorded and an exothermic peak was recorded between −30 and −45 ◦ C. The DSC curve recorded during cooling of the stearin fraction showed two main successive exotherms. Crystallisation was initiated at 27.5 ◦ C with a very sharp and intense peak (from 27.5 to 25 ◦ C), then a broad exothermic peak was recorded between 18.5 and −23 ◦ C. Regarding the thermal characteristics, the initial temperature of crystallisation recorded for milk fat, Tc = 20 ◦ C, was intermediate between the olein fraction (Tc = −8.5 ◦ C), and

Fig. 2. Differential scanning calorimetry curves recorded during cooling of milk fat, stearin fraction and olein fraction from 70 to −50 ◦ C at |dT/dt| = 1 ◦ C min−1 .

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the stearin fraction (Tc = +7.5 ◦ C). Thus, dry fractionation of milk fat modified its thermal properties: the initial temperature of crystallisation, as well as the thermal profile were changed. 3.3. Crystalline structures formed on cooling at |dT/dt| = 1 ◦ C min−1 3.3.1. Crystallisation properties of milk fat Fig. 3 shows the 3D plots of the XRD patterns recorded at small and wide (inset) angles as a function of time during cooling of milk fat at |dT/dt| = 1 ◦ C min−1 . They corresponded to the longitudinal organisation of TG molecules and to the lateral packing of the acylglycerol chains, respectively. The formation of a single ˚ −1 (4.16 A), ˚ recorded at peak of diffraction at q = 1.51 A wide angles during cooling of milk fat, corresponded to crystallisation of TG molecules in an hexagonal chain

Fig. 3. Structural evolution of milk fat as a function of temperature. Three-dimensional plots of small and wide (inset)-angle X-ray diffraction patterns recorded during cooling of milk fat at |dT/dt| = 1 ◦ C min−1 from 60 to −7 ◦ C. The types of longitudinal double (2L) and triple (3L) chain lengths are labeled at small angles for each of the reflexions (Miller index, h k l, with l = 1, 2, 3, 4 or 5) and the peak characterising the ␣ lateral packing is noted at wide angles. q: scattering vector; T: temperature.

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packing, which is the most unstable polymorphic form (␣ form). The bump observed at higher temperature was due to X-ray diffusion and corresponded to the organisation of the fatty acid chains in their liquid state (Larsson, 1972). The successive formation of three longitudinal organisations of TG molecules was recorded at small angles from 22 ◦ C. They corresponded to three different types of lamellar structures, e.g. crystals, formed by TG molecules. The first crystalline structures formed on cooling were double chain length (2L) structures: ˚ (22 ◦ C) and 2L2 = 41.6 A ˚ (21 ◦ C). The XRD 2L1 = 47.3 A peak related to the formation of the 2L1 structure was higher in intensity and better defined than the peak related to the 2L2 structure. From 16 ◦ C, the simultaneous formation of five sharp XRD peaks was related to the crystallisation of TG molecules in a triple chain length ˚ Coupling of (3L) structure with a thickness of 72.1 A. wide and small-angle XRD showed that the three lamellar structures formed on cooling of milk fat corresponded to a chain packing of ␣ type. Fig. 4 shows the evolution of maximal intensity as a function of temperature of the XRD peaks recorded at small angles during cooling of milk fat (Fig. 3) and the corresponding DSC recording. The evolution of the maximal intensity of each small-angle XRD peak plotted as a function of temperature clearly showed the successive crystallisation of the three different lamellar structures formed during cooling of milk fat (Fig. 4). The XRD peak related to the 2L1 structure rapidly increased in intensity from 22 to about 18 ◦ C and reached a plateau until the end of the experiments, meaning that no more TG molecules incorporated in this structure. The intensity of the XRD peak related to the formation of the second 2L structure greatly increased from 21 to about 15 ◦ C, then continued to increase until the end of the cooling process. The simultaneous increase in intensity of the four sharp peaks recorded from 16 ◦ C confirmed that they were related to the same crystalline structure: ˚ The simultaneous XRDT and DSC experi3L = 72.1 A. ments allowed us to relate the structural and thermal data. Thus, the first exotherm recorded by DSC corresponded to crystallisation of TG molecules in the two bilayered ˚ and 2L2 = 41.6 A. ˚ The second structures, 2L1 = 47.3 A exotherm formed on cooling of milk fat was related ˚ to crystallisation of the trilayered structure, 3L = 72.1 A (Fig. 4B). Thus, the crystallisation properties of milk fat on cooling at |dT/dt| = 1 ◦ C min−1 can be summarised as follows: ˚ ) + ␣ 2L2 (41.6 A ˚ ) Liquid 1 → ␣ 2L1 (47.3 A ˚ ) + liquid 2. + ␣ 3L (72.1 A

Fig. 4. Analysis of small-angle X-ray diffraction peaks as a function of temperature and differential scanning calorimetry (DSC) recording during cooling of milk fat at |dT/dt| = 1 ◦ C min−1 . (A) Evolution of the maximal intensities of the diffraction peaks recorded in Fig. 3 which correspond to long spacings (100% corresponds to the strongest peak); (B) DSC crystallisation curve recorded simultaneously from the same sample. 2L: bilayered stacking; 3L: trilayered stacking; ␣: hexagonal packing.

3.3.2. Crystallisation properties of stearin fraction Fig. 5 shows the 3D plots of the XRD patterns recorded at small and wide (inset) angles as a function of time during cooling of stearin fraction at |dT/dt| = 1 ◦ C min−1 . The XRD patterns recorded at small angles showed the formation of three different types of crystals during cooling of stearin fraction. The first crystals of TG that were formed from 26 ◦ C ˚ and corresponded to two 2L structures, 2L1 = 47.5 A ˚ 2L2 = 41.7 A. Then, two broad peaks were formed from 13 ◦ C. They corresponded to the third type of crystals formed on cooling, which was a 3L structure with a ˚ Comparing the area under the smallthickness of 68 A. angle XRD peaks, the results showed that the main crystalline structures formed on cooling of stearin fraction at 1 ◦ C min−1 were 2L longitudinal organisation of TG molecules. The low XRD intensity of the first order

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Fig. 5. Structural evolution of stearin fraction as a function of temperature. Three-dimensional plots of small and wide (inset)-angle X-ray diffraction patterns recorded during cooling of stearin fraction at |dT/dt| = 1 ◦ C min−1 from 60 to −7 ◦ C. The types of double (2L) and triple (3L) chain length stackings are labeled at small angles for each of the reflexions (Miller index, h k l, with l = 1 or 2) and the peaks characterising the ␣ and ␤ lateral packings are noted at wide angles. q: scattering vector; T: temperature.

of diffraction of the 3L structure (3L0 0 1 peak) corresponded to crystallisation of TG present in low amount. The wide-angle XRD patterns showed from 26 ◦ C the ˚ characterformation of a single peak centred at 4.16 A, istic of crystallisation in the ␣ form. Then, from 22 ◦ C, two peaks corresponding to crystallisation in the ␤ form ˚ The ␣ were simultaneously recorded at 3.85 and 4.3 A.  and ␤ polymorphic forms coexisted until the end of the experiment, and their respective intensities increased as a function of the decrease in temperature during cooling of stearin fraction. Fig. 6 shows the evolution of maximal intensity of the XRD peaks recorded a small angles as a function of temperature during cooling of stearin fraction. The ˚ and 2L2 = 41.7 A, ˚ were two 2L structures, 2L1 = 47.5 A ◦ simultaneously formed at 26 C during cooling of stearin fraction. However, their behaviour as a function of time was different. The 2L1 structure highly increased in intensity in the 26–20 ◦ C T-range, then a plateau was observed until the end of the experiment at −7 ◦ C. The 2L2 structure progressively increased in intensity from

25

Fig. 6. Analysis of small-angle X-ray diffraction peaks as a function of temperature and differential scanning calorimetry (DSC) recording during cooling of stearin fraction at |dT/dt| = 1 ◦ C min−1 . (A) Evolution of the maximal intensities of the small-angle diffraction peaks recorded in Fig. 5 (100% corresponds to the strongest peak); (B) DSC crystallisation curve recorded simultaneously from the same sample. 2L: bilayered stacking; 3L: trilayered stacking.

its formation at 26 ◦ C until the end of the experiment. ˚ The XRD peaks related to the formation of the 3L = 68 A ◦ structure, which was formed from 13 C, increased in intensity during cooling. Plotting the evolution of intensity of the small-angle XRD peaks with the DSC signal recorded simultaneously on the same figure allowed us to relate the structural and thermal data. The DSC crystallisation curve recorded on cooling of stearin fraction at 1 ◦ C min−1 showed first the formation of a very sharp and intense exothermic peak and secondly the formation of a large exothermic event. The first exothermic peak with an initial temperature of crystallisation of 27.2 ◦ C was ˚ crystalline related to the formation of the ␣ 2L1 = 47.5 A structure. The two overlapped peaks recorded from about 20 ◦ C were related to the successive crystallisation of the ˚ structure and the 3L = 68 A ˚ structure as a 2L2 = 41.7 A ˚ function of the decrease in temperature. The 2L2 = 41.7 A  structure was associated with the ␤ polymorphic form.

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C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

˚ structure may correspond either to the ␣ The 3L = 68 A  or to the ␤ polymorphic forms, it was not possible to conclude. From the structural analysis performed in this study, it is possible to summarise the crystallisation properties of stearin fraction as follows: ˚ ) + ␤ 2L2 (41.7 A ˚ ) Liquid 1 → ␣ 2L1 (47.5 A ˚ ) + liquid 2. + ␣ or ␤ 3L (68 A 3.3.3. Crystallisation properties of olein fraction Fig. 7 shows the 3D plots of the XRD patterns recorded at small and wide (inset) angles during cooling of olein fraction at |dT/dt| = 1 ◦ C min−1 . The five sharp XRD peaks recorded simultaneously at small angles from 13 ◦ C corresponded to the crystallisation of a 3L ˚ Traces structure characterised by a thickness of 72.1 A.

Fig. 7. Structural evolution of stearin fraction as a function of temperature. Three-dimensional plots of small and wide (inset)-angle X-ray diffraction patterns recorded during cooling of olein fraction at |dT/dt| = 1 ◦ C min−1 from 60 to −7 ◦ C. The types of triple (3L) and double (2L) chain length stackings are labeled at small angles for each of the reflexions (Miller index, h k l, with l = 1, 2, 3, 4 or 5) and the peak characterizing the ␣ lateral packing is noted at wide angles. q: scattering vector; T: temperature.

˚ were recorded at −7 ◦ C, at the of a 2L structure (47.7 A) end of the cooling process. The XRD patterns recorded at wide angles (Fig. 7) showed the formation of a single ˚ corresponding to the crystallisation of the peak at 4.16 A fatty acid chains in a lateral packing of ␣ type. Fig. 8 shows the evolution of maximal intensity of the XRD peaks recorded at small angles as a function of temperature during cooling of olein fraction (Fig. 7) and the DSC curve recorded simultaneously. The five XRD peaks recorded at small angles (Fig. 7) showed a parallel increase in intensity (Fig. 8A). This result confirmed that they were related to the same crystalline organisation, i.e. the 3L structure. Two domains can be delimited considering the evolution of the peak intensities: from 13 to about 5 ◦ C and from 5 to −7 ◦ C. These results showed that crystallisation of olein fraction apparently occurred ˚ strucon a single crystalline structure, the ␣ 3L (72.1 A) ture, but in several steps (in fact, the structural behaviour might be more complex as shown by DSC). The first step

Fig. 8. Analysis of small-angle X-ray diffraction peaks as a function of temperature and differential scanning calorimetry (DSC) recording during cooling of olein fraction at |dT/dt| = 1 ◦ C min−1 . (A) Evolution of the maximal intensities of the diffraction peaks recorded in Fig. 7 which correspond to long spacings (100% corresponds to the strongest peak); (B) DSC crystallisation curve. 3L: trilayered stacking.

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of crystallisation was related to the first exothermic event recorded on cooling, which was constituted by two sharp overlapped peaks. The second step of crystallisation is related to the exothermal bump recorded at about 5 ◦ C and at lower temperatures. On cooling of olein fraction at 1 ◦ C min−1 , one main type of crystals was formed from the liquid phase: ˚ ) + liquid 2. Liquid 1 → ␣ 3L (72.1 A 4. Discussion The crystallisation of milk fat from the melt and the subsequent filtration of the slurry is a common industrial process to obtain milk fat fractions with different chemical and physical properties. Dry fractionation is based on the different thermal (crystallisation and melting) properties of TG resulting from their different fatty acid composition. The physical properties of the fractions will depend on fractionation conditions such as temperature, cooling rate, agitation and efficiency of filtration to a certain extend but also on the initial composition of milk fat (e.g. seasonal variations) (Lavigne, 1995; Kaylegian and Lindsay, 1995). Then, it is worth noting that fractionation is not unique and will slightly vary with conditions used. However, the physical properties of the fractions will depend primarily on crystal types formed initially which are always of 2L type and mainly composed of trisaturated TG. Changing the final crystallisation temperature affects primarily fractionation yield. As expected, the fractions obtained in this study at 21 ◦ C in the conditions detailed above were chemically distinct and had different T-range for crystallisation (Fig. 2). Since the onset temperatures for crystallisation are probably all above the homogeneous nucleation temperature, the number concentration and the types of catalytic impurities present (i.e. diacylglycerols, monoacylglycerols, free fatty acids, phospholipids) might determine the onset temperature (Table 1). The stearin fraction was enriched in long-chain saturated fatty acids that have higher melting points, whereas olein fraction that was liquid at 21 ◦ C, contained principally TG with unsaturated and short-chain fatty acids (Table 2; Fig. 1). This study showed that a natural fat such as milk fat, which is constituted by a mixture of TG molecules with large differences in acyl chain length and degree of unsaturation, leads to the formation of several types of crystals. It was evidenced that TG molecules segregate into two main types of crystals during cooling ˚ and 3L (72.1 A) ˚ lamelof milk fat: 2L (41.6–47.3 A) lar organisations (Fig. 3). This is in agreement with the fact that high-melting molecules first tend to segregate

27

from the remaining liquid and form crystals when fat is cooled (Larsson, 1994). The 2L structures formed at higher temperature may be composed by TG of the stearin fraction, whereas the 3L structure may be formed by TG of the olein fraction. This study showed that even if milk fat consists of many different TG molecules, its crystallisation behaviour corresponds to the TG fraction that dominates the polymorphic behaviour. The crystallisation properties of milk fat observed on cooling at |dT/dt| = 1 ◦ C min−1 were similar to those obtained previously by Lopez et al. (2005). The physical properties of milk fat, including crystallisation behaviour and polymorphism, are dependent not only on the physical and chemical properties of the TG but also on the interaction between the TG. For these reasons, this study was performed to understand how TG structure in both the stearin and olein fractions influence the phase behaviour and polymorphism of milk fat. It is worth noting that the polymorphic species obtained during cooling depend on the cooling rate as shown previously (Lopez et al., 2001a,b, 2002, 2005). The proportions of solid and liquid phase at a given temperature also vary as a function of the crystallisation conditions. To our knowledge there is no technique including NMR, DSC and X-ray able to provide absolute quantification of the solid fat content (SFC). However, to allow comparison of the data provided for the different fractions we determined by direct integration of DSC melting recordings the amount of solid phase at −6 ◦ C. We found SFC of about 97, 94 and 80% for stearin, milk fat and olein, respectively (Lopez et al., in preparation). On cooling of milk fat and its primary fractions, the hexagonal chain packing (␣ form) was formed from the melt. This polymorphic form persisted until the end of the experiment, at −7 ◦ C, for milk fat and olein fraction (Figs. 3 and 7). Concerning crystallisation of stearin fraction, the ␣ form was also formed but in coexistence with a more stable polymorphic form, i.e. the ␤ form. Both small-angle XRD and simultaneous DSC indicated the slower growth rate of the ␤ form compared to that of ␣ form in relation with molecular packing kinetic (Fig. 6). The accommodation of various TG molecules with different chain lengths is apparently easier in the ␣ form in comparison to the ␤ form due to the low lateral density of chains in the former (Wesdorp et al., 2005). This has important consequences visible at XRD peak widths of both types of phases (Fig. 5) that will be examined in the discussion related to liquid crystal formation. Concerning the ␤ form, the plot of line intensity versus temperature (Fig. 6A) showed (i) an increase of growth rate as a function of decreasing temperature and as supercooling increase, (ii) a clear break point in this increasing rate at

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C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

˚ structure developed. the temperature where the 3L (68 A) The fast rate growth of this third species formed during cooling of stearin fraction seems similar to that of ␣ form. When both events can be related and existence of a transition between ␣ and ␤ can be ruled out by the constant value of ␣ form intensity in this temperature domain, it is not clear whether 3L form is ␣ or ␤ . The growth rates observed for ␣ and ␤ forms were opposite as indicated Fig. 6A. The relationship between the two growth rates is also puzzling. While it seems that no monotropic transition ␣ → ␤ exists between both species, the simple Avrami or related models based on single species development proposed for analysis of crystal growth cannot explain the curve shapes of Fig. 6A since two species develop at the same time in non-isothermal conditions (Marangoni, 2005). The study of crystal growth and nucleation during cooling in non-isothermal conditions has been addressed recently by Marangoni et al. (2006). Considering the crystallisation properties of both stearin and olein fractions, the results showed that the structural characteristics of both fractions depended on their TG composition. Double chain length structures were mainly formed ˚ and during cooling of the stearin fraction: ␣ 2L1 (47.5 A) ˚ In the first crystalline structure formed ␤ 2L2 (41.7 A). ˚ the upon cooling of the stearin fraction, ␣ 2L1 (47.5 A), molecules are arranged perpendicular to the methyl endgroup plane and the hydrocarbon chains are hexagonally

loosely packed (Fig. 5). A schematic representation of the packing of the 2L2 structure characterised on cooling of the stearin fraction is proposed in Fig. 9. From a molecular packing point of view, the ␣ form has an irregular methyl end-group region and the main part of the hydrocarbon chains is “oscillating” (Hernqvist, 1988) or display random disorganisation along a common direction perpendicular to the layer plane. The structure of the glycerol groups and the organisation of the methyl end-group are not known. However, the sharpness of the lines and the number of reflections observed (see below) indicates that packing was dominated by glycerol group organisation. The second crystalline structure ˚ A schematic formed on cooling is the ␤ 2L2 (41.7 A). representation of this structure is proposed Fig. 9. The hydrocarbon chains of ␤ form are arranged according to the orthorhombic subcell inducing the tilt of the chains in relation to the organisation of end-group planes. The structural constraints generated by packing of molecules of very different types was shared by all parts of the molecules, glycerol, chains and end-groups. The behaviour of the first 2L structure formed on cool˚ and ing was similar in the stearin fraction (2L1 = 47.5 A) ˚ in milk fat (2L1 = 47.3 A). The 2L1 phase developed in a 4–6 ◦ C range and then stopped growing, in the 26–20 ◦ C T-range for stearin fraction and in the 22–18 ◦ C T-range for milk fat. These crystals may be constituted by TG with long-chain saturated fatty acids which showed the higher melting points. According to Small (1986), a

Fig. 9. Proposed structure of the ␣ form and ␤ form of triglycerides with double chain length longitudinal organisations as seen in the ca projection. For the ␣ form, the methyl end-group regions are somewhat disordered, as in liquid crystals.

C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

trisaturated TG with similar fatty acids (same number of carbon atoms per fatty acid n) forms a 2L structure ˚ n + 4.1 A ˚ with an angle of 90◦ (␣ form). with d = 2.59 A ˚ lamellar structures correThus, the 2L1 = 47.3–47.5 A sponded to a mean fatty acid chain length of about 16.7 atoms of carbon. This 2L1 structure may be constituted by palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids. Thus, TG molecules such as PSS, PPS and MPS or PPO may be incorporated in this 2L1 structure. The 2L2 ˚ which was formed at lower temstructure (41.6–41.7 A) perature developed during the cooling process, meaning that TG molecules were incorporated in this structure until the end of the experiments. It is important to note that this structure corresponded to a hexagonal subcell (␣ form) in milk fat whereas it corresponded to a ␤ form in the stearin fraction. Furthermore, compared to the 2L1 structure, the 2L2 structure developed much more in the stearin fraction compared to milk fat. According to Small (1986), a ␤ 2L structure form a longitudinal organisa˚ n + 4.43 A ˚ (tilt of the chains = 66◦ ). tion with d = 2.32 A Thus, the mean chain length of the 2L2 structure may correspond to 16 atoms of carbon. The TG molecules with different chain length fatty acids, and particularly with one chain in the TG molecule differs in length by at least four carbons (difference in nC ≥ 4; Larsson, 1986), may ˚ structure. It is also possible crystallise in the 3L (68 A) that these kinds of TG were not crystallised at the end of the experiments at −7 ◦ C (Fig. 2). Moreover, efficiency of the fractionation process is limited by the contamination of the stearin fraction by a liquid fraction entrapped within the stearin crystal network (evaluated to about 50 ± 10% of olein left into the stearin, while the solubility of trisaturated TG in olein is about 8–10%), the composition of which corresponds to that of olein. On cooling of olein fraction at 1 ◦ C min−1 , TG molecules self-organised in a triple chain length struc˚ with a hexagonal lateral packing corture, 3L = 72.1 A, responding to ␣ polymorphic form (Fig. 7). Compared to the stearin fraction, an increase in the degree of unsaturation and differences in chain length in the olein fraction induced a lower melting point and altered the polymorphic behaviour of TG molecules. The difference in acyl chain length gave rise to a chain disorder in the methyl end-group region. The ␣ form has vertical chains in relation to the absence of organisation of end-group plane and can accommodate various TG molecules with different chain length and unsaturation in the same crystal. Considering the proportion of the fatty acid chains of the olein fraction, the calculated mean number of atoms of carbon was 15.2. As the carbon–carbon distance in ˚ the zig–zag plan of the acylglycerol chains is 1.27 A (Small, 1986), the mean length of a fatty acid chain was

29

˚ The 3L structure is constituted by 15.2 × 1.27 = 19.3 A. adding the lengths of three fatty acid chains to the sum of two glycerols and one terminal methyl group packing. The length corresponding to the three fatty acid ˚ Thus, the calculated sum of two chains was 57.9 A. glycerols and one terminal methyl end-group packing ˚ This value is in excellent agreewas: 72.1–57.9 = 14.2 A. ˚ for the ␣ 3L ment with Lutton (1948) who found 15.3 A ˚ structure formed by C10C18C18. (73.7 A) The longitudinal organisation of TG molecules is determined by the similarity of the hydrocarbon chains (Lutton, 1972). Triple chain length structures are formed when one fatty acid chain differs much from the other two in length or in saturation (Sato, 1996). Then, the different fatty acid chains segregate into one chain layer and the two remaining chains are located in another layer. The olein fraction used in this study, was enriched in TG with a short-chain fatty acid, i.e. butyric and capro¨ıc acids, and two long-chain fatty acids (Table 2; Fig. 1). However, this short-chain fatty acid may not segregate in one layer and the two other chains in a separate layer as its concentration in the olein fraction was too low to form a single type of crystals structured like that. Olein fraction was enriched in TG with two monounsaturated long-chain fatty acids or two medium-chain fatty acids or TG with one monounsaturated and one medium-chain fatty acids. This kind of TG may govern the structural properties of the olein fraction during cooling, by segregating the long-chain (un)saturated fatty acids. The schematic representation of the ␣ 3L structure ˚ value charwhich is proposed in Fig. 10 explains the 72 A acterised using XRD (Fig. 7). The short and mediumchain length fatty acids are packed in layers A, with a mean number of atoms of carbon of about 13.5, and unsaturated and saturated long-chain fatty acids are in layers B, with a mean number of atoms of carbons of about 18. As no single crystal study exists for the ␣ form, the structures are illustrated only schematically. The chain axes are indicated as lines since the main part of the hydrocarbon chains is supposed randomly defined (oscillating) around a fixed direction perpendicular to that of the planes of the layers giving the 0 0 l XRD lines. Then, molecules are arranged perpendicularly to the methyl end-group plane and the hydrocarbon chains are hexagonally close packed. This proposed TG packing is based on the position and intensities of the small-angle XRD lines corresponding to planes 0 0 l with 1 ≤ l ≤ 5. This 3L stacking has the electronic density profile along direction c shown in bottom right of Fig. 10 with two domains of larger density corresponding to the esters groups separating the fatty acid chains. Such an electronic density profile allowed to obtain the calcu-

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˚ Fig. 10. Proposed triglyceride (TG) packing for the 3L ␣ form of milk fat and its olein fraction. The structure corresponding to the 3L form (3L = 72 A) of small-angle X-ray diffraction (XRD) patterns of Fig. 7 is shown schematically at bottom left as seen in the ca projection. It corresponds to the packing of short and medium-chain length fatty acids in layers A with unsaturated and saturated long-chain fatty acids in layers B. This stacking of TG molecules displays the electron density profile shown at bottom right. The corresponding small-angle XRD pattern calculated with Carine® software is shown in figure (top). The Miller indexes of 0 0 l planes, with l = 1, 2, 3, 4 or 5 are indicated. The aa projection in the chain mean direction shown as an inset displays an hexagonal subcell corresponding to wide-angle XRD patterns in Fig. 7 (inset).

lated small-angle XRD pattern shown in top of Fig. 10. This calculated pattern is similar to that experimentally obtained (Fig. 7). The experimental small-angle XRD pattern corresponding to the 3L structure is characterised by: (i) the presence of important sharp lines for four of the five orders of diffraction observed and (ii) the higher intensity of the second order compared to the first order, (iii) a quasi null diffraction intensity at the fourth order. The sharpness of the single small-angle XRD peak, ˚ structure described corresponding to the ␣ 3L (72.1 A) above, is surprising since it corresponds to crystallisation of the wide diversity of TG molecules contained in the olein fraction (Table 2) (not considering the trace amounts, ≤1%, of 2L structure also observed). The

sharp crystallisation exotherm observed by DSC recording indicated that hundreds of different molecules with different chain lengths and unsaturations were packed quasi-simultaneously. The presence of traces of polar lipids (monoacylglycerols, diacylglycerols) may also play a role in milk fat crystallisation as discussed by Walstra and van Beresteyn (1975) and Foubert et al. (2004). The sharpness of the lines observed by XRD is puzzling. This may be explained by the formation of liquid crystals of smectic type in which the disorder is integrated in the lateral packing of the fatty acid chains. Fig. 11 shows for comparison the small-angle XRD patterns of milk fat, stearin fraction and olein fraction. The milk fat pattern shows XRD lines of the crystalline struc-

C. Lopez et al. / Chemistry and Physics of Lipids 144 (2006) 17–33

31

Table 3 Characteristics of the crystalline structures observed by X-ray diffraction recorded at both small and wide angles during cooling at 1 ◦ C min−1 First crystal

Milk fat Stearin fraction Olein fraction

Second crystal

Third crystal

T (◦ C)

Type

˚ d (A)

T (◦ C)

Type

˚ d (A)

T (◦ C)

Type

˚ d (A)

22 26 13

2L ␣ 2L ␣ 3L ␣

47.3 47.5 72.1

21 26 −7

2L ␣ 2L ␤ 2L ␣

41.6 41.7 47.7

16 13 –

3L ␣ 3L ␣ or ␤ –

72.1 68 –

tures formed in both fractions but does not the sum of them. The crystal structure found in the olein fraction (3L) is found in milk fat while only traces of the structures present in the stearin fraction (2L1 and 2L2 ) were observed. The sharpness of small-angle XRD lines corresponding to ␣ form (3L and 2L1 ) compared to the XRD lines related to the ␤ form (2L2 ) is clearly evidenced (Fig. 11). The evolution of the Peak Width at Middle Height (MHPW) of milk fat and olein fraction are shown as a function of order l as an inset (Fig. 11). It is also worth to note that the MHPW of the small-angle ˚ structure is XRD lines associated with the ␣ 3L (72.1 A) lower in the olein fraction compared to milk fat (Fig. 11, inset). From the MHPW versus l evolution observed for olein fraction, it is possible to deduce using extrapolation ˚ −1 are due to instruof MHPW at l = 0 that about 0.5 A mental broadening of the lines mainly at small-angle position sensitive detector. Then the intrinsic MHPW of the small-angle XRD lines (MHPWi ) only range in ˚ −1 ≤ MHPWi < 0.18 A ˚ −1 for olein while it about 0.04 A −1 ˚ ranges from about 0.12 to 0.2 A for milk fat (Fig. 11,

inset). The further broadening of milk fat ␣ lines is likely due to the presence of defect in the structure due to the presence of more saturated TG foreign molecules. Such sharpness of the lines confort the hypothesis of liquid crystal formation (de Gennes and Prost, 1993). The ␣ form of olein crystals is an interesting model of liquid crystal organisation which is intermediate between the lateral disorganisation of the L ␣ form of phospholipids in which chains are in melted state and that of more stable forms like ␤ that are crystal ordered at the chain level. Such formation of liquid crystals in milk fat and olein fraction should benefit the solubility of aromas. When aromatic molecules are highly soluble in the liquid state of TG, the crystallisation of fat in well-ordered stable crystal is supposed to expel these molecules toward the liquid phase. The presence of liquid crystal and structural defects in milk fat is expected to favour aromatic molecule entrapment. Conversely, oxydability of liquid crystalline fat should be less than that of oil in relation with a lower diffusivity in the former assuming oxygen diffusivity is lower in the crystalline phase than in the oil one (Table 3).

5. Conclusion

Fig. 11. Comparison of the X-ray diffraction patterns of milk fat, stearin fraction and olein fraction recorded at small angles at −7 ◦ C after cooling from 60 ◦ C at |dT/dt| = 1 ◦ C min−1 . The types of triple (3L) and double (2L) chain length stackings are labeled for each of the reflexions (Miller index, h k l, with l = 1, 2, 3, 4 or 5). (Inset) Evolution of the middle-height peak width of the 3L structure characterised for milk fat and olein fraction as a function of the diffraction order.

The use of time-resolved synchrotron radiation XRD coupled to DSC allowed the identification of the crystalline structures formed on cooling of stearin fraction and olein fraction obtained by dry fractionation from the same batch of anhydrous milk fat. In similar thermal conditions, cooling at dT/dt = 1 ◦ C min−1 , the TG composition influenced the behaviour of crystallisation, e.g. longitudinal organisation of TG molecule and the polymorphic form. The liquid phase was in coexistence with one (olein), mainly two (stearin) or three (milk fat) solid ˚ phases. The crystals corresponded to 2L (47.5–41.7 A)  ˚ and 3L (72.1 A) lamellar structures with ␣ or ␣ + ␤ polymorphic forms. Such a study is important from both the scientific and technological points of view to better understand and then control the physical properties of milk fat.

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