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Milk Fat Thermal Properties and Solid Fat Content in Emmental Cheese: A Differential Scanning Calorimetry Study

UMR 1253 Science et Technologie du Lait et de l’Oeuf, INRA-Agrocampus, 65 rue de Saint-Brieuc, 35042 Rennes Cedex, France

¹ Corresponding author: Christelle.Lopez{at}rennes.inra.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The experiments reported in this study give deeper insight into the crystallization of milk fat in Emmental cheese, which is the most widely consumed hard cheese in France. Differential scanning calorimetry (DSC) was used to monitor the thermal properties of milk fat after the main stages involved during manufacture of Emmental cheese. By heating the samples to 60°C to eliminate their thermal history and cooling them at 2°C/min, the liquid solid phase transition of fat was investigated. Confocal laser scanning microscopy was used to characterize in situ the supramolecular organization of milk fat dispersed in the casein matrix. The destabilization of fat globules by aggregation or coalescence and the formation of free fat during the manufacture altered the thermal properties of milk fat by increasing the initial temperature of crystallization and by the formation of 2 overlapping exotherms. The melting properties of the crystalline structures formed by fat at the temperatures used for ripening (12, 21, and 4°C) were examined. Differential scanning calorimetry was used to determine the ratio of solid to liquid fat; that is, the amount of fat that is crystallized, by dividing the partial enthalpy of melting of the fat for ripening temperature by the total enthalpy of melting of the same fat extracted from cheese. This study shows, for the first time, that milk fat is partially crystallized in Emmental cheese: about 55.7 ± 3.5% of fat is solid at 4°C at the end of ripening. Polymorphic phase transitions of milk fat are also suggested during ripening of Emmental cheese.

Key Words: triacylglycerol • polymorphism • fat crystal • confocal laser scanning microscopy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Milk fat determines the specific properties of fat-rich products, such as butter and cream. In food products, fat may be dispersed as oil in water emulsion (as in cream, yogurt, and cheese) or may be the continuous phase (as in butter). Moreover, the fat phase may be crystallized at the temperature of storage and consumption, as is the case for dairy products. Thus, examination of the thermal properties of fat in complex food products and determination of the solid fat content are of particular importance in understanding their rheological, sensorial, and nutritional properties.

Milk fat is composed mainly of triacylglycerols (TG), esters of fatty acids and glycerol, which represent 97 to 98% of total fat. More than 400 fatty acids have been identified, varying in chain length and unsaturation (Gresti et al., 1993; Jensen and Newburg, 1995). The extreme diversity of milk fatty acids and TG, each characterized by its own melting point, induces a wide melting range, which spans from about –40 to 40°C. At intermediate temperature, milk fat is partially crystallized and corresponds to a mixture of solid fat (crystals) and liquid fat (oil).

Fat is dispersed in milk in the form of droplets, called the milk fat globules, which have a volume-weighted diameter of approximately 4 µm. A biological membrane composed mainly of phospholipids and proteins organized as a trilayer envelops the milk fat globules (Keenan and Dylewski, 1995). Due to technological processes, the structure of milk fat globules can be greatly modified and fat can be dispersed in dairy products as coalesced fat globules, aggregates of fat globules, tiny homogenized fat globules, or nonglobular fat (Lopez, 2005a).

The thermal properties of milk fat are usually studied by differential scanning calorimetry (DSC). Differential scanning calorimetry is a useful tool for determining the temperature of final melting and initial crystallization of fat, as well as for following polymorphic evolutions. Studies using DSC have given an insight into the thermodynamics of milk fat and milk fat fraction transitions in bulk (Timms, 1980; Lavigne, 1995; Marangoni and Lencki, 1998; ten Grotenhuis et al., 1999) and in emulsions (Walstra and van Beresteyn, 1975). Differential scanning calorimetry studies monitored on milk fat globules showed that the temperature of the beginning of crystallization is delayed as a function of the decrease of their size (Lopez et al., 2002a; Michalski et al., 2004b). Timms (1980) and Marangoni and Lencki (1998) studied anhydrous milk fat (AMF) by DSC and observed that it crystallizes and melts in several steps. Researchers agree on the existence of 3 overlapping endotherms recorded on heating corresponding to separate groups of TG that melt separately (Timms, 1980; Marangoni and Lencki, 1998). These 3 groups of milk fat TG are called the low melting point (LMP), medium melting point (MMP), and high melting point (HMP) fractions. Lavigne (1995) identified the main TG of each fraction and related them to the thermal and structural properties of the whole milk fat. The complex DSC recordings result from both the broad distribution of TG composition and the polymorphism of TG (Hagemann, 1988). Triglycerides have the ability to crystallize in different polymorphic forms, of which only one is completely stable in given conditions. This polymorphism being mainly monotropic, forms are metastable and the transitions are irreversible. The different polymorphic forms have different compositions, different crystal lattices, and different melting points, which increase with increasing stability (Small, 1986).

Recently, the use of DSC coupled to synchrotron radiation x-ray diffraction allowed identification of the crystalline structures formed by TG molecules as a function of temperature and time in AMF (Lavigne, 1995; Lopez et al., 2001a,b,c) and in milk fat globules (Lopez et al., 2000, 2001c, 2002a,Lopez et al., b, 2005b; Michalski et al., 2004b). Lopez et al. (2001c, 2002a,b) showed that the dispersion state of milk fat; for example, in fat globules or in bulk as AMF, alters both its thermal and structural properties.

Milk fat contributes to the physical properties of dairy products, especially of those with low water content. The functional properties of milk fat are strongly related to its composition and to the amount of fat crystals of various types and sizes that are formed at the temperature of the application. Crystallization of milk fat affects many properties such as 1) its rheological properties, 2) the resistance of fat globules to disruption, and 3) the consistency and mouth feel of high-fat content products. Furthermore, milk fat crystallization may be important for technical applications. Thus, it is of interest to better understand the thermal properties of milk fat in dairy products.

Although DSC is convenient, few studies exist on the thermal properties of fat in complex dairy products, and particularly in cheese. Cheese can be considered as a heterogeneous material, mainly composed of a complex mixture of TG molecules, proteins, and water. Differential scanning calorimetry was used to distinguish natural Mozzarella cheese from imitation Mozzarella made with calcium caseinate (Tunick et al., 1989) and to distinguish Mozzarella cheeses made from cows’ milk and water buffalo milk (Tunick and Malin, 1997). Tunick (1994) examined the effects of homogenization on the melting profiles of cheese fat and free oil in Mozzarella cheese. Results from DSC experiments also showed that the melting profile of milk fat in Mozzarella cheese changes during storage (Rowney et al., 1998). Famelart et al. (2002) and Michalski et al. (2004a) used DSC to investigate the melting behavior of Emmental cheese.

The objective of this study was to investigate the thermal properties of the fat phase dispersed in cheese as a function of time and temperature during both its manufacture and ripening. Among cheeses, Emmental is of particular interest as it is successively ripened at different temperatures; that is, 12, 21, and 4°C. Differential scanning calorimetry was used to characterize in situ the crystallization and melting of TG molecules considering the supramolecular organization of fat during the manufacture of Emmental cheese. The melting properties and polymorphic evolutions of fat were characterized and the solid fat content was determined during the ripening of Emmental cheese.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Samples
Emmental Cheese Manufacture and Ripening.
Three separate trials of Emmental cheese were manufactured, from separate milks, using a specialized pilot plant based on the research center of INRA, Rennes (Chalon Megard, La Cluse, France).

Day –1.
Raw whole milk, purchased from a local dairy plant (Triballat, Noyal-sur-Vilaine, France), was collected the day before Emmental production (d –1). The milk was adjusted to a fat content:total N ratio of 0.86 using skimmed milk, and then stored overnight at 4°C.

Day 0.
Standardized milk was pasteurized using an Actijoule pilot pasteurizer (Actini, Evian les Bains, France) at 63°C for 20 s. The cheese milk (850 L) was heated to 31°C in a temperature-controlled milk vat. A lysozyme solution (Delvosyme, Gist Brocades, Seclin, France) was added to milk at a concentration of 0.01 mL/kg. The milk was supplemented with 0.01 mL/kg of a 510 g/L CaCl₂ solution. Milk was inoculated as follows: lactococci (0.4 units/850 kg; EZAL MM100, Rhodia, Dangé-Saint-Romain, France); thermophilic lactobacilli (LH 100, Rhodia) inoculated in the milk at 0.47 mL/kg; thermophilic streptococci (PAL ITG ST 82-87, Standa, France) inoculated in the milk at 1.65 mL/kg, propionic bacteria (0.05 g/kg; PAL ITG P9, Standa). After a 30-min incubation at 31°C, milk pH was adjusted to 6.62 using CO₂ dissolved directly in milk. Calf rennet was added at 0.25 mL/kg (145, Berthelot, ABIA S.A. Meursault, France). Curd was formed after 22 min and hardened for 4 to 5 min (20% of the clotting time). The curd was cut and curd grains were mixed and heated for 20 min to a temperature of 51°C. Curd was drained off under vacuum (30 kPa) in the racking unit and molded at 47°C in a 780-mm diameter and 27-mm height mold (Doryl, France). The curd was pressed for 4 h at 0.4 kPa (room temperature = 24°C). After pressing, the curd was turned over and acidified for 19 h in a temperature-controlled room at 24°C.

Day +1.
The curd was demolded (pH 5.17 ± 0.03), weighed, and placed in a cold brine bath for 48 h (saturated NaCl solution: 350 g/L; 12°C; pH 5.2).

Day +1 to 52.
Cheese was ripened in a temperate room for 16 d at 12°C, 85% relative humidity, and then in a warm room for 28 d at 21°C, 80% relative humidity, and finally in a cold room for 7 d at 4°C, for a total ripening time of 52 d. Some Emmental cheeses were kept for 7 d more at 4°C. Three Emmental cheeses of about 80 kg each were manufactured in total.

AMF Extracted from Emmental Cheese.
Anhydrous milk fat was extracted from Emmental cheese 1) in the 12°C room, 2) in the 21°C room, 3) in the 4°C room, and 4) at the end of ripening, as detailed below.

Cream and AMF.
Creams (fat content ~320 g/kg) were obtained by skimming fresh raw milk collected in Brittany and purchased from a local dairy plant (Triballat, Noyal-sur-Vilaine, France). The creams were collected during the same season and the same year as cheese milk used to manufacture Emmental cheese. Anhydrous milk fat was extracted from creams as detailed below.

Physicochemical Analysis
Determination of Fat Content and DM.
Fat content was determined using the SBR (Schmid, Bondzynski, Ratzlaff) method (IDF/FIL, 1986). Dry matter was measured by drying 2 g of cheese mixed with sand at 102°C ( ± 2°C) for 7 h (IDF/FIL, 1987). Table 1 shows the fat content and DM of the samples characterized after the main stages involved during the manufacture of Emmental cheese. The DM increased due to loss of whey after cutting and heating of curd grains and as a consequence of pressing. The DM slightly increased during the ripening period indicating continuous loss of moisture. The increase in DM is responsible for the increase in fat concentration during the manufacture of Emmental cheese. At the end of ripening, the composition of the cheeses was within specified limits for Emmental (IDF/FIL, 1981).

Preparation of Methyl Esters.
In a screw-capped tube, a 5-mL fat solution in hexane was prepared at a concentration of 10 mg/mL. Then, 50 µL of sodium methoxide (Sigma-Aldrich, Saint Quentin Fallavier, France), a solution of internal standard of glycerol tripelargonate (Sigma-Aldrich) stored at –20°C under nitrogen, and a trace of anhydrous sodium sulfate were added. The reagents were incubated for 15 min at room temperature, and centrifuged at 1,000 x g at 20°C for 10 min (SV-11-TH model, Firlabo, France). The upper layer was recovered and diluted 10 times in hexane before injection into the gas chromatograph (Ulberth et al., 1999). Fatty acid methyl esters were measured on a Varian gas chromatograph (model 3800, Walnut Creek, CA) equipped with a flame-ionization detector, a programmed temperature injector, and a capillary column (50 m x 0.33 mm; film thickness 0.25 µm) coated with 70% cyanopropyl polysilphenylene-siloxane (BPX-70, SGE, Ringwood, Victoria, Australia). Experimental conditions were as follows: initial temperature of on-column injection (1 µL) was 40°C for 0.2 min; the temperature injector was then programmed to increase to 200°C at a rate of 200°C/min; an isotherm at 200°C for 6 min; and a decrease in temperature to 40°C at a rate of 200°C/min. Detector temperature was 250°C; carrier gas was hydrogen at a pressure of 24.1 kPa. Oven temperature was programmed as follows: 40°C for 10 min followed by an increase to 205°C at a rate of 5°C/min; oven was maintained at 205°C for 20 min.

Microstructural Analysis
Emmental cheese microstructure was examined after the main stages involved during its manufacture using confocal laser scanning microscopy (CLSM). Thin slices of cheese, measuring approximately 5 x 5 x 3 mm thick, were prepared from the freshly cut samples, using a scalpel. The protein network was stained using acridine orange fluorescent dye (Aldrich Chemical Company, Inc., Milwawkee, WI). A lipid-soluble Nile Red fluorescent dye (Sigma-Aldrich, St Louis, MO) was used to label fat. About 0.5 mL of each staining solution was put on the coverslip. Then, the coverslip was placed on the slice of cheese to permit the diffusion of the stains for 30 min in the dark at 4°C. Microstructural analysis was performed using a confocal Leica TCS NT microscope (Leica Microsystems, Heidelberg, Germany), which used an argon/krypton laser in dual-beam fluorescent mode, with excitation wavelengths of 568 and 488 nm for fat and protein, respectively. The two-dimensional images had a resolution of 1,024 x 1,024 pixels, and the pixel scale values were converted into micrometers using a scaling factor. In the double-stained samples, the fat phase was colored red and the protein phase was colored green; the aqueous phase appeared as black areas in the confocal micrographs.

Particle Size Measurements
The fat globule size distributions in Emmental samples and cream were measured by laser light scattering using a Mastersizer 2000 (Malvern Instruments, Malvern, UK), equipped with a He/Ne laser (wavelength = 633 nm) and an electroluminescent diode (wavelength = 466 nm).

To determine the fat globule size in the curd and curd grains, 1 g of the sample was dissociated with 5 mL of dissociation buffer [6 mol/L urea, 100 mmol/L EDTA, 20 mmol/L imidazole buffer, pH 6.6 (99%, Prolabo, Fontenay sous Bois, France)], and stirred for 30 min at room temperature before measurement (Dalgleish, 1984). The fat globule size distribution in cream was measured after dispersion in distilled water.

The refractive index of milk fat was taken to be 1.460 at 466 nm and 1.458 at 633 nm as previously determined in our laboratory (Michalski et al., 2001). All analyses were performed in triplicate. From the size distribution, the average volume-weighted diameter, d₄₃ = n_i/n_i (where n_i is the number of fat globules in a size class of diameter d_i), was calculated by the instrument software.

Thermal Properties
The thermal properties of the samples were monitored by DSC using a TA Q-1000 calorimeter (TA Instruments, New Castle, DE). Calibration was made with an indium standard, melting point = 156.66°C, H melting = 28.41 J/g.

Emmental Cheese.
About 50 to 70 mg of cheese was accurately weighed in a hermetic stainless steel pan of 100 µL. An empty pan was used as a reference, and both pans were hermetically sealed. Two protocols were used to study the thermal properties of Emmental cheese. In the first protocol, the structures formed after elimination of the thermal history were characterized. The samples were heated to 60°C to melt all existing nuclei, cooled from 60 to –5°C at 2°C/min, and heated from –5 to 60°C at 2°C/min. In the second protocol, the structures formed at the temperature of ripening were characterized. The samples of cheese were conditioned at the temperature of ripening, T_ripening = 12, 21, and 4°C, and then transported in an isothermal box to the calorimeter precooled to T_ripening. The samples of cheese were tempered for 5 min at the chosen temperature. Then, the cooling and heating kinetics were monitored as follows: 1) From T_ripening (12, 21, 4°C) to 60°C at 2°C/min; 2) From T_ripening (12, 21, 4°C) to –5°C at 2°C/min, then from –5 to 60°C at 2°C/min. Measurements were performed in triplicate with independent cheese samples.

Cream.
About 5 to 10 mg of cream was weighed in a hermetic aluminium pan of 50 µL. An empty hermetic aluminium pan was used as reference, and the pans were hermetically sealed. The creams were 1) heated to 60°C, 2) cooled from 60 to –5°C at 2°C/min; and 3) heated from 5 to 60°C at 2°C/min. Measurements were performed in triplicate with independent creams.

AMF.
About 5 to 10 mg of AMF extracted from Emmental cheese samples and cream were weighed in a hermetic aluminium pan of 50 µL. An empty hermetic aluminium pan was used as reference. The pans were hermetically sealed. Two protocols were used as follows. In the first protocol, AMF samples extracted from cream were heated to 60°C to melt all existing nuclei, cooled from 60 to –5°C at 2°C/min, and heated from –5 to 60°C at 2°C/min. In the second protocol, AMF samples extracted from Emmental cheeses were heated to 60°C to melt all nuclei, cooled from 60 to –40°C at 2°C/min, and heated from –40 to 60°C at 2°C/min. Measurements were performed in triplicate with independent AMF samples.

The temperature of the beginning of crystallization (T_onset, in °C), of final melting (T_offset, in °C), and the enthalpy of melting (H expressed in J/g of sample) were calculated by the software (Advantage Software version 2, TA Instrument). Because the enthalpy of melting of TG is proportional to the amount of fat in the samples, the results were divided by the concentration of fat determined for each sample.

Statistical Analyses
Analyses of variance were performed using the GLM procedure of Statgraphics Plus version 5 (Statistical Graphic Corp., Englewood Cliffs, NJ) to determine 1) the effect of the stage of manufacture of Emmental cheese on the concentration of fatty acids and on the thermal parameters (T_onset, T_offset), and, 2) the effect of the stage of ripening on the thermal parameters (H, T_offset) and on the calculated solid fat content. Differences between the treatment means were compared at the 5% level of significance using the Fisher’s least significance difference (LSD) test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Fatty Acid Composition
The fatty acid composition of the samples analyzed during the manufacture of Emmental cheese is given in Table 2. The results showed that the fatty acid composition did not evolve significantly (P > 0.05) during the manufacture of Emmental cheese; that is, from the formation of the curd until after brining.

View larger version (16K): [in this window] [in a new window]   Figure 1. Differential scanning calorimetry curves recorded during cooling at|dT/dt|= 2°C/min, from 60 to –5°C, of rennet-induced curd (A), heated curd grains before pressing (B), and Emmental cheese after pressing (C), at 1 d (D), after brining (E), and at the end of ripening (52 d; F). Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

The thermal properties of fat recorded on cooling changed during the manufacture of Emmental cheese. Thus, the DSC crystallization curves were delimited in 3 temperature ranges or domains as indicated in Figure 1. The first domain delimited on cooling corresponded to TG molecules in their liquid state. The second domain corresponded to the liquid solid phase transition with the formation of the first exothermic peak. The third domain corresponded to the formation of the second exothermic peak. The 2 exotherms recorded on cooling were related to crystallization of 2 independent groups of TG molecules (Lopez et al., 2002a).

Figure 2 shows the DSC curves recorded during heating from –5 to 60°C at 2°C/min of the samples previously cooled in the calorimeter (Figure 1). All the DSC melting curves showed 1) an endothermic peak from the beginning of the experiment to about 15°C, 2) a second endothermic event from about 15 to 20°C, and 3) an endothermic event with at least 2 overlapped peaks, from about 20°C to the final melting temperature of the samples, where the DSC curve reached the baseline. For the DSC melting curves corresponding to Emmental cheese after brining (Figure 2E) and at 52 d (Figure 2F), an exotherm was clearly recorded, between 13 and 16°C, between the first and the second endothermic peaks recorded on heating. The overlapped peaks, endothermic and exothermic, showed that several crystalline reorganizations occurred during heating.

View larger version (19K): [in this window] [in a new window]   Figure 2. Differential scanning calorimetry curves recorded during heating at 2°C/min, from –5 to 60°C, of rennet-induced curd (A), heated curd grains before pressing (B), and Emmental cheese after pressing (C), at 1 d (D), after brining (E), and at the end of ripening (52 d; F), after cooling at 2°C/min from 60 to –5°C (Figure 1). Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

View larger version (12K): [in this window] [in a new window]   Figure 3. Differential scanning calorimetry curves recorded during cooling of cream and anhydrous milk fat extracted from the cream, from 60 to –5°C at 2°C/min. Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

View larger version (13K): [in this window] [in a new window]   Figure 4. Differential scanning calorimetry curves recorded during heating of cream and anhydrous milk fat extracted from the cream, from –5 to 60°C at 2°C/min, after cooling from 60 to –5°C at 2°C/min. Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

View larger version (158K): [in this window] [in a new window]   Figure 5. Confocal laser scanning micrographs of Emmental cheese taken after the main stages involved during manufacture: A) rennet-induced curd, B) heated curd grain before pressing, C) Emmental cheese after pressing, D) Emmental cheese after brining. Fat is colored red; proteins are colored green; the serum phase is black.

View larger version (10K): [in this window] [in a new window]   Figure 6. Fat globule size distribution measured after dissociation of the casein matrix of () rennet-induced curd, () heated curd grains, and () Emmental cheese after pressing.

View larger version (12K): [in this window] [in a new window]   Figure 7. Differential scanning calorimetry melting curves recorded upon heating of Emmental cheese from the temperature of ripening to 60°C at 2°C/min: A) after 16 d at Tripening = 12°C; B) after 28 d at Tripening = 21°C; C) after 7 d at Tripening = 4°C; and D) Emmental cheese at the end of ripening, stored at 4°C. Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

The melting properties of milk fat globules concentrated in a cream (fat content ~320 g/kg) were investigated for comparison with the melting properties of cheese fat during ripening of Emmental. The DSC melting curve of the cream stored 7 d at 4°C showed an endothermic event composed of overlapping peaks with a maximum at 21.8°C and a final melting temperature of 40.9 ± 0.1°C (Figure 8A). Figure 8B shows the DSC melting curve of the cream first heated to 60°C to eliminate its thermal history and kept at room temperature (i.e., 19 to 21°C) during 8 h. The endothermic peak recorded on heating, from 26 to 41°C with a maximum at 32.2°C, showed that some fat crystals were formed in fat globules at room temperature.

View larger version (11K): [in this window] [in a new window]   Figure 8. Differential scanning calorimetry melting curves recorded on heating at 2°C/min of milk fat globules concentrated in a cream stored for A) 7 d at 4°C, and B) 8 h at 20°C. Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

The amount of fat that was crystallized after 16 d in the 12°C room (44.08 ± 5.73%), 28 d in the 21°C room (18.76 ± 2.03%), and 7 d in the 4°C room (53.35 ± 4.52%) were significantly different (P < 0.001). A longer storage of Emmental cheese at 4°C after the end of ripening did not significantly (P > 0.05) increase the amount of solid fat (Table 4).

The solid fat content within Emmental cheese stored at 4°C after its ripening process was calculated as a function of temperature by constructing an integral curve from the DSC melting thermogram (Figure 9). This curve shows that 38% fat is crystallized in Emmental cheese at 20°C, 22% is crystallized at 25°C, and that 3% is crystallized at the mouth temperature (37°C).

View larger version (28K): [in this window] [in a new window]   Figure 9. Evolution of the solid fat content as a function of temperature determined in Emmental cheese at the end of ripening (52 d), using differential scanning calorimetry.

View larger version (20K): [in this window] [in a new window]   Figure 10. Differential scanning calorimetry melting curves recorded during heating of Emmental cheese from –5 to 60°C at 2°C/min, following different kinetics of cooling; 1) after cooling from the temperature of ripening (Tripening) to –5°C at 2°C/min (solid line), and 2) after heating cheese to 60°C to eliminate the thermal history and cooling from 60 to –5°C at 2°C/min (dashed line). The melting properties of Emmental cheese were determined after the main stages involved during its ripening: A) Tripening = 12°C, B) Tripening = 21°C; the insert shows the crystallization curve recorded on cooling from 21 to –5°C; C) Tripening = 4°C; and D) Emmental cheese at the end of ripening, stored at 4°C. Heat flow is given in arbitrary units; Endo > indicates the direction of the endothermic reaction.

The DSC melting curves recorded after elimination of the thermal history of Emmental cheese showed similar shapes and thermal events (Figure 10). They were constituted by overlapped peaks: a first endotherm between –5 and 13 to 14°C, an exotherm between 14 and 16°C, a second endotherm between 16 and 19 to 20°C, and a broad endothermic event from about 20°C to the final melting temperature of fat in Emmental cheese.

Comparing the DSC melting curves recorded in the same range of temperature; that is, from –5 to 60°C, and at the same heating rate, e.g., dT/dt = 2°C/min, differences due to the thermal history of Emmental cheeses were observed (Figure 10; Table 5). The melting peaks and suggested polymorphic transitions were different (Figure 10). Furthermore, the crystalline structures formed by fat during ripening of Emmental cheese had a significantly (P < 0.05) higher final melting temperature (To_ffset) than the same fat after elimination of its thermal history (Table 5). The differences in final temperature of melting between unstable and more stable crystalline species were in the range 1.72 to 2.82°C (Table 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

Quantification of Milk Fat Crystals in Emmental Cheese Using DSC
In this study, the amount of fat crystals was determined by calculating the ratio of solid to liquid fat (solid fat content) using DSC for the 3 temperatures used in the ripening process of Emmental cheese (Table 4). Solid fat content values were derived from DSC melting curves by sequential integration. The calculation of the solid fat content involved the implicit assumption that the heat of melting of TG was constant. In fact, the heat of melting had a positive correlation with melting point and, as a result, the true proportion of solids was overestimated (by approximately 7 to 10%) at high solid fat contents (Norris and Taylor, 1977). From a methodological point of view, the determination of H(total cheese fat) is difficult. The better procedure would be to determine H_{(total cheese fat)} directly in Emmental cheese. However, this is not possible because the water present in cheese (DM = 627 g/kg) crystallizes below –5°C. Thus, upon heating in the –40 to 60°C range, the H melting of water is superimposed on the H melting of fat in cheese, hindering the determination of H_{(total cheese fat)}. Thus, to approximate H_{(total cheese fat)}, cheese fat was extracted from cheese and analyzed in bulk (AMF). The authors consider that both the dispersion state of fat and the metastability of crystalline species may influence the estimation of H_total AMF: 1) H_total is higher in bulk than in the dispersed state (5 to 10%); that is, in Emmental cheese. Considering the dispersion state of fat in Emmental cheese, H_total AMF is overestimated; 2) H_total AMF recorded on heating is lower after cooling at 2°C/min (formation of unstable species) than after storage for a long period at different temperatures during ripening of cheese (stable species). Considering the metastability of the crystalline species formed, H_total is underestimated. The difference in H between stable and unstable polymorphic forms is in the range from 10 to 20%. Thus, the authors consider that the H_total AMF used to calculate the solid fat content in Emmental cheese is underestimated to about 10 to 15%.

Differential scanning calorimetry allowed us to quantify the amount of fat that was crystallized as a function of temperature until its final melting, and to characterize the thermal properties and probably polymorphism of fat upon cooling and heating in a complex and high fat content food product. The method developed, which had never been used before to quantify the solid fat content in food products, is innovative. For the first time, to the author’s knowledge, the solid fat content was determined in a cheese and during the ripening process of Emmental cheese.

Nuclear magnetic resonance (NMR) spectroscopy is usually used to quantify the solid fat content of fats and oils. Most NMR studies of dairy products have focused on AMF or blends of AMF and vegetable oils (Lambelet, 1983b; Marangoni et al., 2000; Singh et al., 2002). In contrast, only few NMR relaxation measurements on fatty products with intermediate water content have been carried out (Samuelsson and Vikelsoe, 1971; Chaland et al., 2000; Wiking et al., 2005). Nuclear magnetic resonance spectroscopy is not yet adapted for determining the solid fat content in complex products in which the fat is dispersed. Moreover, the polymorphism of milk fat increases the difficulty of interpreting the NMR data. Recently, Bertram et al., (2005) determined the crystallization temperatures of milk fat globules using ¹H NMR transverse (T₂) relaxation measurements during continuous cooling of creams. The latter authors confirmed that both the chemical composition and the supramolecular structure of fat (i.e., size of fat globules) may contribute to the T₂ relaxation pattern and showed, for the first time, that phase transitions (i.e., fat crystallization) in cream can be determined using NMR transverse relaxation measurements.

Differential scanning calorimetry and NMR were compared for determination of the solid fat content in anhydrous fats in stable polymorphic forms (Lambelet, 1983a, b). Solid fat content values derived from DSC curves were higher than values obtained by NMR spectroscopy. The latter author attributed these results to differences in the chemical composition of the fats and concluded that results obtained by the NMR method were too low. Furthermore, the differences between DSC and NMR solid fat contents are not constant, but show variations as a function of temperature (Norris and Taylor, 1977). The values of solid fat content determined using NMR found in the literature are lower than the solid fat content determined in the current study using DSC (Table 4). Wiking et al. (2005) determined that after 50 min at 4°C, 27.8 to 44.2% of fat was solid in cream, depending on the fatty acid composition. Samuelsson and Vikelsoe (1971) calculated that after 72 h storage at 12°C, about 38% of fat was crystallized in cream.

The presence of fat crystals in Emmental cheese (solid fat content ~55 to 56% at 4°C) was observed in this study, but precise localization of fat crystals within the inclusions of fat dispersed in Emmental cheese was not investigated. The determination of the size and the localization of such fat crystals, which may be dispersed in the interior of the fat inclusions or may be at the interface with the protein network, could provide some understanding of the flavor development and flavor release in complex matrices such as cheeses. Indeed, some aroma compounds are solubilized in the liquid phase of fat and their release depends on the ratio of solid to liquid fat (Relkin et al., 2004). Furthermore, the melting of fat crystals may contribute to the release of these aroma compounds and to mouthfeel during consumption of Emmental cheese.

We showed in this study that the final melting point of fat in Emmental cheese was 40.76 ± 0.25°C (Table 5), which means that the fat was totally liquid only over this temperature. Thus, fat crystals do not play a role in the functional properties— flowability, melt-ability, and stretchability—of melted Emmental cheese. Furthermore, the amount of fat that was solid above 37°C was calculated to be 3% (wt/wt; Figure 9). This fraction of solid fat can be mainly constituted by high-melting point TG; that is, TG with saturated long-chain fatty acids such as palmitic acid (C16:0). Regarding the digestive and nutritional properties of milk fat, studies found in the literature did not consider that milk fat is partially solid for temperatures >37°C. The mechanisms involved in the hydrolysis of such solid TG molecules by the digestive lipolytic enzymes have not been investigated and thus are not well known.

Importance of the Supramolecular Organization of Fat on Thermal Properties
The crystallization and melting properties of milk fat changed during the manufacture of Emmental cheese (Figures 1 and 2; Table 3). The experiments, which were performed using DSC at the same cooling rate (2°C/min) showed the formation of a second exothermic peak at higher temperature during the manufacture. The crystallization properties of fat may depend on 1) the composition of fat because the fatty acid composition of milk fat determines the temperature at which it begins to crystallize (Shi et al., 2001); 2) the dispersion of fat (Lopez et al., 2002a); that is, its supramolecular organization, or 3) accumulation of lipolysis products during ripening (Foubert et al., 2004). The fatty acid analysis did not show any significant (P > 0.05) changes in the composition of fat as a function of time and mechanical treatments during the manufacture of Emmental cheese (Table 2). Regarding the microstructural analysis, the CLSM micrographs showed changes in situ in the supramolecular organization of fat during the manufacture of Emmental cheese (Figure 5). Furthermore, the DSC experiments performed using DSC on cream and AMF indicated the importance of the supramolecular organization of fat, fat globules vs. bulk, on its thermal properties (Figures 3 and 4). The size distribution of fat globules in cream was similar to the size distribution of fat globules entrapped in the curd (~4.5 µm). Thus, the delay in the temperature of the beginning of crystallization observed in the curd, T_onset = 17.61 –15.34 = 2.27°C, may be due to the lower content of fat (lower signal to noise ratio) or explained by the environment of fat; that is, caseins and water. Lopez et al. (2002a) showed the formation of 2 overlapped exotherms during cooling of cream at 1 and 3°C/min. The differences observed may be related to the size of fat globules in the creams; the latter authors used a cream with larger fat globules (up to 30 µm). Smaller fat globules show a single exotherm on cooling (Lopez et al., 2002a). Regarding our results obtained on cooling of AMF, we agree with the existence of 2 exothermic processes that correspond to crystallization of 2 groups of TG (Lavigne, 1995; ten Grotenhuis et al., 1999).

Comparing cream and AMF, initial crystallization was initiated at different temperatures and in a different way, as previously observed (Walstra and van Beresteyn, 1975; Söderberg et al., 1989; Lopez et al., 2001c, 2002a,b). Initial temperature of crystallization recorded for cream (T_onset = 17.61°C) was slightly lower than that of AMF (T_onset = 18.03°C). The initial crystallization of fat was related to supercooling relaxation and then to the nucleation rate in the dispersed state (Walstra and van Beresteyn, 1975). The slopes of the initial peak of crystallization were 0.016 arbitrary units (a.u.)/°C for cream and 0.407 a.u./°C for AMF. The sharp peak was related to a higher crystal growth rate for AMF compared with individual milk fat globules of cream. Although crystal growth and material supply for growing, which occurred by molecular diffusion, were necessarily limited in fat globules, it was not so in a far greater volume of bulk fat. Considering the influence of the size of milk fat globules on their crystallization properties, Lopez et al. (2002a) showed that reducing the size of milk fat emulsion droplets by homogenization induces a higher supercooling; that is, the initial crystallization temperature was displaced toward the lower temperature. The same results were obtained with native milk fat globules of different sizes (Michalski et al., 2004b). The differences in supercooling were generally explained by the theory of nucleation for crystallization (Walstra and van Beresteyn, 1975). When the liquid fat is dispersed in emulsion droplets, at least one nucleus must be formed in every droplet to achieve full crystallization. This implies that a lower temperature was needed for a finer dispersion because more catalytic impurities were needed. Thus, the time needed to obtain the first nuclei was inversely proportional to fat globule volume (Walstra and van Beresteyn, 1975).

The DSC recordings of Emmental cheese corresponded to a sum of the phenomenon that occurred during cooling and heating of fat dispersed in a continuous protein network. The increase of fat globule size by coalescence and the formation of free fat after pressing of Emmental cheese could explain the higher temperature of crystallization due to lower supercooling, and the faster crystal growth rate related to the first exothermic peak recorded on cooling (Figure 1).

The DSC melting curves corresponding to Emmental cheese after elimination of their thermal history were similar to those recorded for AMF (Figures 2 and 4). The DSC melting curves recorded on heating of cream and AMF were similar to the results obtained by Lopez et al. (2002a, 2005c), using coupled DSC and x-ray diffraction techniques. It has been shown that polymorphic transitions are facilitated in AMF by a comparison with cream (Lopez et al., 2002b). Thus, the exothermic processes recorded with Emmental cheese after brining and at the end of ripening could be related to monotropic transitions on heating favored by the presence of nonglobular fat in the protein matrix.

Phase Transition of Milk Fat Fractions in Emmental Cheese During Ripening
The experiments performed in this study showed that 44.08 ± 5.73% of the fat was crystallized after 16 d of ripening in the 12°C room (Table 4). Then, the increase in temperature from 12 to 21°C in the warm room ( _ripening = +9°C) induced the melting of some TG crystallized at 12°C, which lead to the increase in the amount of liquid TG from about 66 to about 81 to 82%. This liquid phase could have favored structural rearrangements of TG molecules in relation to the monotropic polymorphism of TG, because mixed crystals can dissolve and transform into a new and more stable polymorphic form (Small, 1986). Furthermore, T_ripening = 21°C decreased the viscosity of liquid fat compared with 12°C and could have promoted fractionation of the fat. Figure 7B shows the DSC curve of the high-melting point peak formed in Emmental cheese, which corresponded to the melting of 18 to 19% fat (Table 4). The cooling of the Emmental cheese from 21 to –5°C showed the formation of an exothermic peak corresponding to crystallization of TG molecules in the cheese (Figure 10B insert). The subsequent heating of the sample showed 2 well-separated endothermic peaks, which means that the TG molecules that had crystallized on cooling melted separately on the subsequent heating (Figure 10B). Thus, 2 independent groups of TG were formed in Emmental cheese. The increase in the final melting temperature of TG molecules from 39.9 ± 0.5°C (12°C room) to 40.9 ± 0.2°C (21°C room) showed that crystalline structures with a higher compactness and thus, stability were formed in the warm room because of segregation of TG that form high-melting point crystals; that is, TG with saturated long-chain fatty acids. During 28 d of storage at 21°C, TG molecules had the time to incorporate into a crystal lattice, indicating that the polymorphism of TG depended on kinetic parameters. The storage of Emmental cheese in the 4°C room, T_ripening = –17°C, lead to an increase of the solid fat content from about 19% to about 53 to 54% (Table 4). The DSC melting curve showed the 3 endothermic peaks corresponding to LMP, MMP, and HMP fractions, as described by Timms (1980). The HMP fraction corresponded to the high-melting point peak formed for T_ripening = 21°C (Figure 7). The formation of the LMP and MMP peaks suggested that polymorphic evolutions of TG molecules occurred in Emmental cheese during storage at 4°C. The polymorphic transitions that occurred in the solid phase as a function of time were favored and accelerated by the amount of liquid fat phase in Emmental cheese at the temperature of ripening; that is, 12, 21, and then 4°C. The DSC melting curve of the cream stored 7 d at 4°C (Figure 8A) showed a different shape compared with the DSC curve recorded after storage of Emmental cheese 7 d with T_ripening = 4°C (Figure 7C). Thus, the thermal history influenced the types of crystals formed and the melting properties of fat. This result confirmed that storage of Emmental cheese at 21°C before storage at 4°C lead to the segregation of high-melting point TG during the ripening process. Regarding the DSC melting curve of the cream stored at room temperature (Figure 8B), similar results were obtained for Emmental cheese stored in the 21°C room. Thus, the melting behavior of TG molecules crystallized in isothermal conditions at room temperature was similar in a complex matrix such as cheese and in fat globules.

On heating of Emmental cheese stored in the successive temperature-controlled rooms, no exothermic process was recorded (Figure 7). This result showed that there was no rearrangement of TG molecules through polymorphic transition to increase their stability on heating. Thus only the melting of stable crystals of TG molecules was observed. Only structural analysis, achieved with synchrotron radiation x-ray diffraction, would allow the identification of the molecular arrangement of TG molecules in the crystalline structures formed and to relate them to the thermal events. The DSC melting curves recorded for Emmental cheese in the 4°C room and at the end of ripening were similar to those of Famelart et al. (2002) and to the results obtained in Lopez et al. (2002b) for cream and AMF after 135 and 105 h of storage at 4°C, respectively. Lopez et al. (2002b) previously showed that the fat phase of cream and AMF is partially crystallized during long storage at 4°C and displays a complex polymorphism that is time and temperature dependent.

	CONCLUSIONS

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The examination of the physical properties of fat in complex food products, such as cheese, is of particular importance. The increased knowledge of the thermal properties and crystallization of fat might be of value to understand the functional and sensorial properties and for technical applications.

To the authors’ knowledge, this is the first time that a dynamic study is performed as a function of time and temperature to characterize the thermal properties of fat and to estimate the solid fat content, in situ in cheese. It has been shown that the supramolecular organization of fat dispersed in Emmental cheese (e.g., fat globules, aggregates of fat globules, or free fat) alters both its crystallization and melting properties. Furthermore, the temperature of ripening greatly influences the solid fat content and the type of crystals present in Emmental cheese. This study showed, using DSC, that 55.7 ± 3.5% of the fat phase dispersed in Emmental cheese is crystallized at the end of ripening at 4°C. As may be the case in many foods, the fat phase is partially crystallized at temperatures below 41°C. Thus, fat inclusions and fat globules are a mixture of crystals and oil between their storage temperature and 41°C. Furthermore, the present study suggested that the fat phase displays a complex polymorphism within a water-dispersed and highly complex processed food product such as Emmental cheese.

	ACKNOWLEDGEMENTS

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The authors thank the technical assistance of R. Primault for CLSM experiments (Microscopy Department, University Rennes I) and E. Beaucher for chemical analysis. M.-H. Famelart (INRA-STLO, Rennes) is acknowledged for her help in statistical analysis. The authors gratefully acknowledge M. Ollivon (CNRS, Châtenay-Malabry) for valuable scientific discussions.

Received for publication November 15, 2005. Accepted for publication March 10, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


Ali, A. R., and P. S. Dimick. 1994. Melting and solidification characteristics of confectionary fats: Anhydrous milk fat, cocoa butter and palm kernel stearin blends. J. Am. Oil Chem. Soc. 71:803–806.

Bertram, H. C., L. Wiking, J. H. Nielsen, and H. J. Andersen. 2005. Direct measurement of phase transitions in milk fat during cooling of cream –