# Milk fat globule fractions recovered by disc stack centrifugal separation of camel milk: Process-induced fat balance, microstructure and lipid composition as a function of size

Imène Felfoul, Christelle Lopez

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Imène Felfoul, Christelle Lopez. Milk fat globule fractions recovered by disc stack centrifugal separation of camel milk: Process-induced fat balance, microstructure and lipid composition as a function of size. LWT - Food Science and Technology, 2025, 215, pp.117223. 10.1016/j.lwt.2024.117223. hal-05263431

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# Milk fat globule fractions recovered by disc stack centrifugal separation of camel milk: Process-induced fat balance, microstructure and lipid composition as a function of size

Imène Felfoul<sup>a,b</sup>, Christelle Lopez<sup>b,c,*</sup>

## ARTICLE INFO

Keywords:

Dromedary milk

Milk processing

Centrifugation

Biological membrane

Milk polar lipid

Milk fat globule size

## ABSTRACT

In recent years, camel milk has become the focus of growing interest in different parts of the world due to its nutritional significance, sustainability, and promising opportunities for dairy product processing. The objectives of this study were (i) to increase knowledge about the size-based fractionation of camel milk fat globules (CMFGs) occurring during conventional centrifugation with a cream separator, (ii) to calculate the fat balance, (iii) to determine the fatty acid, cholesterol and polar lipid compositions as a function of CMFGs size, and (iv) to examine the microstructure of the CMFGs membrane. A comparative analysis with bovine milk was performed. Centrifugal separation of camel milks induced the recovery of CMFGs-enriched creams (317.8 ± 12.1 g/kg of fat; mean diameter: 3.2 µm), and of semi-skim milks containing 16.7 ± 1.2 g/kg of fat dispersed in small CMFGs of 0.5 µm in diameter. The fat balance revealed that a high amount of camel milk fat was recovered in the aqueous phase, i.e. 47 %wt versus 53 %wt recovered in the cream. Small CMFGs contained a greater amount of membrane polar lipids than larger ones: 37.4 ± 2.4 versus 13.6 ± 0.4 mg/g fat. CMFGs contained 41 g sphingomyelin per 100g polar lipids, corresponding to 15.3 ± 0.8 mg/g fat in small CMFGs and 7.4 ± 0.7 mg/g fat in whole milks, which is of nutritional interest. The microscopic observations of the CMFGs membrane revealed a heterogeneous distribution of proteins and polar lipids with circular lipid microdomains rich in sphingomyelin and possibly cholesterol. This study opens perspectives for the valorization of the semi-skim milk fraction recovered as a co-product of camel cream preparation.

## 1. Introduction

Milks from various mammal species (e.g., cow, sheep, goat, buffalo, yack, camel) and dairy products (e.g., creams, cheeses) obtained by milk processing and transformation are consumed worldwide as essential foods in the human diet, particularly for infants and children (FAO, 2021). They are important dietary sources of calcium, proteins and lipids (Jensen, 1995). Among these nutrients, milk lipids are of special interest. The dietary lipids provided by milk have a specific composition compared to other sources of dietary lipids (e.g., vegetable oils), a unique organization into multiscale assemblies referred to as milk fat globules, techno-functional properties and important nutritional and health functions that are far from being totally elucidated (Lopez, 2020a; Lopez et al., 2019).

Bovine milk has been the subject of many scientific research studies in comparison with the milks from other mammal species since it is the milk that is the most extensively produced in terms of volume, the most widely available and consumed worldwide, and the most frequently transformed into dairy products (FAOSTAT, 2024). However, in recent years, non-bovine milk has been the focus of increasing interest. The opportunities for the production of non-bovine milks and their transformation into dairy products have expanded since they can be used to develop specialized dairy products with high value-added of regional and cultural, as well as international importance, for human nutrition in all age groups. Non-bovine milks such as camel milk are therefore of great interest to people as well as to the dairy sector.

The number of camels worldwide is estimated at about 41.77 million in 2022 (FAOSTAT, 2024), 89% of which have one hump (Camelus

<footnote><sup>a</sup> University of Sfax, LAVASA, ENIS, Sfax, Tunisia</footnote>

<footnote><sup>b</sup> INRAE, STLO, F-35000, Rennes, France</footnote>

<footnote><sup>c</sup> INRAE, BIA, F-44316, Nantes, France</footnote>

<footnote>* Corresponding author. INRAE, UR BIA, 3 impasse Yvette Cauchois, La Géraudière CS 71627, F-44316, Nantes, France.</footnote>

<footnote>E-mail address: christelle.lopez@inrae.fr (C. Lopez).</footnote>
dromedarius) and are located in North Africa, West Asia and Australia, and the other 11% of which have two humps (Camelus bactrianus) and are mainly distributed in central Asian countries, including China and Mongolia (Faye, 2020). Camels are important semi-ruminant animals that produce milk with the following mean gross chemical composition: 3.5% fat, 3.1% protein, 4.4% lactose, 0.8% ash and 12% total solids (Alhaj & AlKanhal, 2010). Camel milk has been traditionally consumed by humans for thousands of years for its excellent nutritional value and various bioactivities, as a rich source of antimicrobial and antioxidant substances, as well as for its medicinal value and health benefits (Al-Juboori et al., 2013; Berhe et al., 2017; Mihic et al., 2016). Camels have recently become the subject of increasing scientific and commercial interest since climate change and future environmental challenges including water and feed resources availability are already influencing traditional cow-based cattle productivity in various parts of the world. The camel is an animal with exceptional physiological characteristics that requires low amount of water and feed resource, and that is capable of surviving in hostile climatic conditions with elevated temperatures (Berhe et al., 2017; Seyiti et al., 2024). The production of camel milk is promoted as a strategy of sustainable adaptation to drought due to lack of rain induced by climate change, e.g., in east of Africa. Being widely regarded as a sustainable and promising livestock species for the development of health-beneficial foods or ingredients, camel milk consumption is gradually becoming popular in China among urban consumers due to large population and rapid economic development (Seyiti et al., 2024). The launch of new camel dairy products is an emerging trend. In recent years, the growing consumption of dairy products made from camel milk (cheese, cream, yogurt, fermented milk, ice-cream, butter, ghee) has required more knowledge in order to adapt camel milk processing (Mtibaa et al., 2021).

In milk produced by all mammal species, lipids are dispersed in the form of spherical droplets referred to as milk fat globules. Milk fat globules are unique delivery systems for biologically-active molecules in the gastrointestinal tract. They share a similar structure with a core rich in triacylglycerols (TAG; 95% of dairy lipids) and are surrounded, protected and stabilized by a biological membrane called the milk fat globule membrane (MFGM) (Lopez, 2020b). However, milk fat globules secreted from various mammal species exhibit different sizes, profiles and abundances of lipids and membrane proteins, and their properties have not yet been fully investigated. The diameter of milk fat globules naturally extends from 0.2 to 15 µm (Singh, 2006). Some variations in the mean diameters of milk fat globules exist as a function of the mammal species producing the milk. Camel milk exhibits the smallest milk fat globule size distribution (El-Agamy, 2017; El-Zeini, 2006; Walter et al., 2020). Furthermore, camel milk fat is characterized by a large amount of small milk fat globules containing a large amount of polar lipids in their biological membrane (Attia, Kherouatou, Fakhfakh, Khorchani, & Trigui, 2000; Karray et al., 2005a; Mehaia, 1995).

It is now well established that there can be differences in the lipid composition (i.e., the internal TAG core and the MFGM) between small and large bovine milk fat globules. Also, authors reported the effect of native milk fat globule size on the physical properties, functionalities and sensory qualities of dairy products (Truong et al., 2016). Furthermore, small and large milk fat globules are likely to behave differently in the gastrointestinal tract (Berton et al., 2012; Meena et al., 2014), possibly affecting the kinetics of digestion and the bioavailability of nutrients. Differences in the lipid composition and properties of native bovine milk fat globules has led to considerable research around the development of procedures for their size-based fractionation from bovine milk using various technological methods such as microfiltration (Lopez et al., 2011), gravity-based separation (Ma & Barbano, 2000), or centrifugal separation using modified two-stages cream separators (Dhungana et al., 2017). However, to date, the cost and low efficiency of many of these processes applied to bovine milk have limited their industrial application. Concerning camel milk, authors reported losses of small milk fat globules in the aqueous fraction after lab-scale

centrifugation (3500×g for 30 min at 4 °C (Walter et al., 2020),) and centrifugal separation performed with conventional cream separators (Kashaninejad & Razavi, 2020; Mtibaa et al., 2021). This means that conventional cream separators that are widely used for camel dairy products preparation (e.g., cream and butter) could fractionate milk fat globules according to their size. Furthermore, these losses of fat in the aqueous fraction negatively affects the functional and nutritional properties of camel dairy products. However, the process-induced and size-based fractionation of camel milk fat globules occurring during conventional centrifugal separation remains poorly characterized. Camel milk fat globules have been the subject of relatively few studies compared to bovine milk fat globules (Karray et al., 2006). Differences in the fatty acid composition and polar lipid composition of camel milk fat globules according to their size remain to be investigated. Thus, the complete characterization of camel milk fat globules according to their size will contribute to a better understanding of their properties and is the first step needed to link both the composition and the structural properties of milk fat globules to their physiological functions.

The objectives of this work were therefore: (i) to analyze the process-induced fractionation of camel milk fat globules that occurs during centrifugal separation using a conventional cream separator and to compare it with bovine milk; (ii) to determine the lipid composition of camel milk fat globules as a function of their size; and (iii) to examine the microstructure of the camel MFGM with adapted fluorescent dyes able to simultaneous localize the polar lipids, glycoproteins and glycolipids.

## 2. Materials and methods

### 2.1. Milk samples

The camel (Camelus dromedarius) milks used in this study were obtained from two different countries located in Africa, i.e., Mauritania and Tunisia. The milks from Mauritania were collected from a camel dairy farm located in the peri-urban area of Nouakchott (Trarza region, southwest Mauritania). The pooled milk samples used were collected from 50 different healthy camels between 2 and 8 months of lactation in sterile milking cans. The dairy plant, Tiviski Camel Milk Dairy (Nouakchott, Mauritania), used camel milk to prepare creams (35 %wt fat, pH 6.7) with an industrial-scale cream separator. The milk from Tunisia was collected from reared camels at an experimental station located in the south (Gabès governorate, Tunisia). The milk used consisted of pooled milk samples assembled from 20 different healthy camels between 2 and 12 months of lactation and collected in sterile milking cans. After milking, the milk samples were stored on ice (temperature: 4 ± 2 °C) and immediately transported by car to the local university laboratories within 4 h, and by plane to the laboratory in France within 24 h using ice boxes (positive temperature to avoid the alteration of camel milk fat globules).

Bovine milks corresponding to bulk tank raw whole milks were produced in the Brittany region of France and provided by the local dairy plant Entremont (Montauban de Bretagne, France).

After milking, sodium azide (NaN₃, 0.02 g/mL) was added to camel and bovine milks to prevent the multiplication of bacteria. The milk samples were stored at room temperature (19 ± 1 °C) for at least 30 min before particle size measurements and confocal microscopy observations. Milk samples were freeze-dried and then stored at -20 °C before chemical analysis.

### 2.2. Centrifugal separation

Centrifugal separation of camel milks and bovine milks was performed with a disc stack centrifugal separator to fractionate the milk fat globule rich cream and the aqueous phase.
### 2.2.1. Principle of centrifugal separation

The basic principle behind a disc stack centrifugal separator is the separation of different components in a mixture based on their difference in density (e.g., oil droplets and water) and size. This is achieved by spinning a bowl containing the mixture at high speed, which generates a centrifugal force. The velocity at which the spherical particles are separated from the fluid in the separator is calculated using Stokes' law: $v = [\Delta\rho (2r^2) (R\omega^2)]/9\mu$ (equation (1)),

where $\mu$ is the dynamic viscosity expressed in Pa x sec or kg/m-sec units, $\Delta\rho$ is the density difference between the density of the particles expressed in kg/m³ and the density of the fluid expressed in kg/m³, r is the radius of the spherical particles expressed in m, the term $R\omega^2$ is related to the centrifuge (R is the distance between the particle and the axis of rotation of the centrifuge expressed in m, and $\omega$ is the angular speed of the centrifuge expressed in rpm or radians/sec), v is expressed in m/sec. In the dairy industry, the disc stack centrifugal separation is widely used to separate bovine milk components by centrifugal force into a milk fat globule-rich cream and a high-density aqueous phase generally referred as skim milk that contains a very small amount of fat (below 1.5 g/kg for bovine skim milk).

### 2.2.2. Specifications

The technological experiments were conducted in the dairy pilot plant of INRAE STLO (Rennes, France). The centrifugal separation of camel and bovine milks was performed with an electric disc stack centrifugal separator ref M09.10000 Elecrem1-Metal (Fourniture laitière, 01370 St Etienne-du-bois, France) referred to as a cream separator in the manuscript. The technical characteristics of the cream separator were as follows: feed rate of 125 L/h, rotational speed of 8000 rpm, 19 separating discs with a diameter of 95 mm each. About 20 kg of camel milk from Mauritania were heated to 60 °C, i.e., above the final melting point of camel milk fat of 44–46 °C (Karray et al., 2005b). As expected with the cream separator, the camel cream was collected on one side, whereas the camel milk high-density aqueous phase referred to as the aqueous fraction in the manuscript was collected on the other side due to differences in density. For comparison, about 20 kg of bovine milk were fractionated with the cream separator in similar technological conditions. The samples were kept at room temperature (19 ± 1 °C) prior to particle size and zeta potential measurements and microscopic observations, which were performed on the same day. The samples were stored at –20 °C before lipid analysis.

## 2.3. Lipid analysis

### 2.3.1. Chemicals, reagents and polar lipid standards

Methanol (HPLC grade) and chloroform stabilized with ethanol (for analysis) were supplied by Carlo Erba Reagents (Val de Reuil, France). Triethylamine (purity >99%) and formic acid (purity >98%) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The standards of glycerophospholipids were purchased from Sigma-Aldrich: L-α-phosphatidylethanolamine dipalmitoyl, N,N-dimethyl (C16:0) (PE; purity 99%), L-α phosphatidylinositol ammonium salt from soybean (PI; purity 98%), 1,2-diacyl-sn-glycero-3-phosphoserin (PS; purity 98%), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC; purity 99%). Milk sphingomyelin (SM; purity 99%) was supplied by Avanti Polar Lipids (Alabaster, AL, USA).

### 2.3.2. Extraction of total lipids and analysis of lipids

An adapted protocol of the cold extraction procedure developed by Folch et al. (1957) was used for the extraction of total lipids from the lyophilized camel and bovine milks, as detailed in Lopez et al. (2014). About 2 g of milk samples were blended with chloroform:methanol (2:1, vol:vol) using a mixer (IKA, Fisher Bioblock). The extract was shaken and equilibrated with one-fourth its volume of a saline solution (NaCl 0.73 g/100g). The upper phase was washed three times with a mixture of 40 mL chloroform:methanol (2:1, vol:vol) and 10 mL NaCl 0.58

g/100g. The solvent phases containing total lipids were filtered (Whatman filter paper, 2.5 µm, Grosseron, France) through a pinch of anhydrous sodium sulfate and evaporated under vacuum. The extraction of total lipids was performed in duplicate or in triplicate to obtain a coefficient of variation below 5%. The total lipids extracted were stored at –20 °C until further gas chromatography (GC) and high performance liquid chromatography (HPLC) analysis. The amount of cholesterol in the milks was determined by GC (ISO 12228-1:2014 EN, 2015). HPLC combined with an evaporative light scattering detector (ELSD) was used for the determination of the polar lipid classes and for the quantification of total polar lipids, e.g., glycerophospholipids and sphingomyelin. The chromatographic method used for the separation of milk polar lipids was previously described by Lopez et al. (2014). Glycerophospholipids and sphingomyelin separations were performed on an Agilent HPLC chain (HP 1100, Agilent, Massy, France) with four solvent lines, a degasser and a quaternary pump. The detector was an Evaporative Light Scattering Detector (ELSD; PL-ELS1000, Polymer Laboratories, Marseille, France). HPChem software (Agilent, Massy, France) permitted the acquisition of data from the ELSD detector. A silica column, 150 × 3 mm with a 3 µm particle diameter (AIT, Houilles, France), and a precolumn in silica with the same packing and internal diameter were used. Dried and filtered compressed air was used as the nebulizing gas at a flow rate of 1.7 L/min and a temperature of 50 °C. The evaporation temperature was 85 °C. The elution program was (i) isocratic conditions with chloroform/methanol/buffer (1 M formic acid, neutralized to pH 3 with triethylamine) 87.5:12:0.5 (vol/vol/vol) from t = 0 min to t = 7 min; followed by (ii) a linear gradient of chloroform/methanol/buffer 87.5:12:0.5 (vol/vol/vol) to 28:60:12 (vol/vol/vol) from t = 7 min to t = 27 min. The mobile phase was brought back to the initial conditions, i. e., 87.5:12:0.5 (vol/vol/vol), from t = 27 min to t = 29 min, and the column was allowed to equilibrate until the next injection at t = 36 min. The total chromatographic run time was 36 min per sample, which consisted of (i) 7 min in isocratic conditions to elute nonpolar lipids; (ii) the linear gradient to elute the polar lipids; (iii) 2 min to restore the initial conditions; and (iv) the last 7 min to re-equilibrate the column. The flow rate was maintained at 0.5 mL/min. The column was maintained at 40 °C. The injection volume was 10 µL per sample. The lipids extracted were weighed (100 mg) and dissolved in 500 µL of chloroform/methanol (88:12, vol/vol) and transferred to capped test tubes for HPLC analysis. Each sample was injected three times.

The identification of individual polar lipids, the glycerophospholipids (PE, PI, PS, and PC) and the sphingomyelin, was carried out by a comparison with the retention time of pure standards, as previously described (Lopez, 2024; Lopez et al., 2008). The quantitative determination of the glycerophospholipids and sphingomyelin was achieved using five calibration curves corresponding to PE, PI, PS, PC and SM. The calibration curves were determined from the area values obtained by injecting 10 µL of chloroform/methanol (88:12, vol/vol) serial diluted solutions of PE (0.1–1.5 µg), PC (0.5–2.5 µg), PS (0.1–1.75 µg), PI (0.1–1.75 µg) and SM (0.5–3 µg). Each solution was prepared and injected in triplicate. Calibration curves were calculated by applying the equations of the power model to the area and concentration values. The sum of glycerophospholipids (PE, PI, PS, and PC) and sphingomyelin concentration was considered to be the total polar lipid concentration.

## 2.4. Particle size measurements

Dynamic light scattering was used to measure milk fat globule size distributions of milk samples with a Mastersizer 2000 particle analyzer (Malvern Instruments, Malvern, UK), equipped with an electroluminescent diode ($\lambda$ = 466 nm) and a He/Ne laser ($\lambda$ = 633 nm). The refractive indices of milk fat were assumed to be 1.460 at 466 nm and 1.458 at 633 nm. A volume of milk corresponding to about 0.2 mL of milk was directly diluted in 100 mL of water in the measurement cell of the apparatus in order to reach 10% obscuration. In order to dissociate the casein micelles, 1 mL of 35 mM EDTA/NaOH, pH 7 buffer was added
to the milk sample. The characteristic parameters were calculated by the software from the size distributions of milk fat globules: the volume-surface average diameter $D_{32}$ (expressed in µm) defined as $\Sigma n_i d_i^3 / \Sigma n_i d_i^2$ (equation (2)) and the volume-weighted average diameter $D_{43}$ (expressed in µm) defined as $(\Sigma n_i d_i^4 / \Sigma n_i d_i^3)$ (equation (3)) where $n_i$ is the number of fat globules of diameter $d_i$ expressed in µm and the modal diameter (expressed in µm) that corresponds to the population of fat globules with highest frequency in volume. The specific surface area (expressed in $m^2/g$ fat) was calculated by the software as $S = 6\phi/D_{32}$ (equation (4)) where $\phi$ is the volume fraction of milk fat expressed and $D_{32}$ is expressed in µm. The experiments were performed in triplicate for each milk.

## 2.5. Zeta potential measurements

To measure the zeta potential of camel and bovine milk fat globules, the samples were prepared by suspending 4–15 µL milk in 10 mL of a buffer (20 mM imidazole, 50 mM NaCl, 5 mM CaCl₂, pH 7.0; $\eta = 0.89$ cp, $\epsilon = 79$). Measurements of zeta potential were performed at 20 °C using a Zetasizer 3000 HS (Malvern Instruments), which was equipped with palladium electrodes and an avalanche photodiode detector. All analyses were performed three times for each milk sample.

## 2.6. Confocal laser scanning microscopy

An inverted microscope, NIKON Eclipse-TE2000-C1si (NIKON, Champigny sur Marne, France), allowing confocal laser scanning microscopy (CLSM) was used. Confocal experiments were performed using an He-Ne laser operating at 543 nm wavelength excitation (emission was detected between 565 and 615 nm) and an argon laser operating at 488 nm excitation wavelength (emission was detected between 500 and 530 nm). The microstructural analysis of camel milk fat globules was performed as previously reported for bovine milk fat globules (Lopez et al., 2010). The staining protocols of milk fat globules and MFGM were developed in our laboratory and previously described (Lopez et al., 2010). The triacylglycerols located in the core of milk fat globules were stained with the lipid-soluble Nile red fluorescent probe (5H-Benzo α-phenoxazine-5-one, 9-diethylamino; Sigma-Aldrich, St Louis, MO, USA), prepared with a concentration of 42 µg/mL. About 10 µL of the Nile red solution was added to 0.5 mL of milk. The fluid matrix of phospholipids in the MFGM was labeled with the fluorescent probe N-(Lissamine rhodamine B sulfonyl) dioleoylphosphatidylethanolamine (Rh-DOPE; Avanti Polar Lipids, Inc., Birmingham, England). About 10 µL of Rh-DOPE was added to 0.5 mL of milk. To localize the carbohydrate moieties in the MFGM, the lectin wheat germ agglutinin, Alexa fluor 488 (WGA; Invitrogen, Cergy Pontoise, France), was used. A 1 mg/mL solution of WGA was prepared by dissolving the powder in 0.2 M PBS buffer, pH 7.4 (Sigma P4417, Sigma-Aldrich, St Louis, MO, USA). All the solutions containing the fluorescent probes were kept frozen until utilization. After the labeling step, the samples were kept at room temperature for at least 30 min before the microstructural analysis. Then, 3 µL of the milk stained with the fluorescent probes was deposited on the glass and observed under the microscope. The observations were performed using a × 100 (NA 1.4) oil immersion objective. The microstructural analyses were performed at room temperature (19 ± 1 °C). The two-dimensional images had a resolution of 512 × 512 pixels and the pixel scale values were converted into micrometers using a scaling factor. In the multiple labeled samples, different colors were used to locate the fluorescent probes.

## 2.7. Statistical analysis

The size and zeta potential measurements and the chemical analyses were repeated independently at least three times. The results are presented as mean values and standard deviations. Analyses of variance (ANOVA) were performed using the General Linear Model procedure of

Statgraphics Plus version 5 (Statistical Graphics Corp., Englewood Cliffs, NJ, USA). Differences between the treatment means were compared at the 5% level of significance using Fisher's least significance difference (LSD) test.

# 3. Results and discussion

## 3.1. Camel milks: fat content, fat globule size distribution and zeta potential

The fat contents as well as the milk fat globule size distributions and zeta potential values in the camel milks collected in Mauritania and Tunisia were determined and compared to bovine milks collected in France.

The mean fat content of the milks collected in Mauritania was 32.4 ± 1.5 g/kg (n = 5 milks; collected from February to June), while the mean fat content of the milks from Tunisia was 31.2 ± 2.1 g/kg (n = 3 milks; collected in June). Overall, fat contents were significantly ($P < 0.001$) lower in the camel milks than in the bovine milks: 32.0 ± 2.3 vs. 39.1 ± 2.1 g/kg milk, respectively. Fat contents were in accordance with values reported in the literature for camel milk (Al haj & Al Kanhal, 2010; Mtibaa et al., 2021; Walter et al., 2020). Fat contents of camel milk range between 1.2 and 6.4 g/100g milk with an average of 3.5 ± 1.0 g/100g milk (Al haj & Al Kanhal, 2010). Bovine milk fat content was also in agreement with the literature, including variations due to season and cow diet (Lopez et al., 2014). Despite the high variations in fat content that can occur in camel milk, e.g., due to high temperature and water restriction, fat content is consistently lower in camel milk than in bovine milk (Al haj & Al Kanhal, 2010).

The size of milk fat globules was significantly lower in the camel milks compared to the bovine milks (Fig. 1). The size distributions of camel milk fat globules were similar in the milks from Mauritania and Tunisia (Fig. 1). Camel milk fat globules ranged from volume mean diameters 0.02–6 µm, with two main populations: small fat globules in the range of 0.02–0.8 µm, and a population of large fat globules in the range of 1–6 µm with a modal diameter of 2.5 ± 0.3 µm (Fig. 1). The mean diameters were similar for the camel milk fat globules in the milks originating from Mauritania and Tunisia. The mean diameters calculated for the camel milks were $D_{32} = 0.5 \pm 0.3$ µm and $D_{43} = 2.2 \pm 0.4$ µm, respectively, in accordance with the literature (Walter et al., 2020). The high proportion of camel milk fat globules smaller than 1 µm has been highlighted by Attia, Kherouatou, Fakhfakh, Khorchani, and Trigui (2000). However, other authors reported a monomodal size distribution ranging from 0.8 to 10 µm for Australian camel milk (Walter et al., 2020). In bovine milk, the size distribution ranged from 0.02 to 15 µm with fat globules smaller than 1 µm and large fat globules with a modal diameter of 4.1 ± 0.2 µm (Fig. 1), in accordance with previous reports (Lopez et al., 2014). The mean diameters calculated from the whole size distributions were smaller for camel milk fat globules compared to bovine milk fat globules (Fig. 1-B). This size distribution of milk fat globules results from the well-regulated biological mechanisms of assembly and extrusion of intracellular lipid micro-droplets through the apical plasma membrane of the epithelial cells of the mammary gland (Heid & Keenan, 2005). As a result of fat globule size, the surface area of the MFGM surrounding fat globules in camel milk was 2-fold higher than for bovine milk fat globules: 14.2–13.4 ± 2.3 vs. 6.3 ± 0.8 $m^2/g$ fat, respectively. This is an important feature of camel milk since the MFGM contains complex proteins and polar lipids that are reported to have nutritional, immunological, neurological and digestive functions (Thum et al., 2021). Furthermore, the MFGM is a key interface between the TAG core and the aqueous environment where microorganisms and digestive enzymes can adsorb (Douellou et al., 2017; Kosmerl et al., 2021; Pafumi et al., 2002). The observations performed at the microscopic level by confocal microscopy with a specific labeling of the milk fat globule core confirmed the smaller size of camel milk fat globules compared to bovine milk fat globules (Fig. 1-C). A positive relationship between milk
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Differences in size between camel and bovine milk fat globules: (A) Representative size distributions of milk fat globules in camel milks collected in Mauritania and Tunisia and bovine milks from France determined by laser light scattering. (B) Mean diameters and specific surface area of fat globules in camel milks from Mauritania (orange) and Tunisia (yellow) and in bovine milks (green). (C) Confocal laser scanning microscopy images of the milks with Nile red as fluorescent dye (C-1: camel milk; C-2: bovine milk). (D) Zeta potential values of fat globules in camel milks from Mauritania (orange) and Tunisia (yellow) and in bovine milks (green). Results correspond to means and standard deviation (n = 9); (****) P < 0.01, NS = non significant P > 0.05; P-values calculated by ANOVA.</figcaption></figure>

fat content and milk fat globule size was previously reported (Lopez et al., 2014; Ménard et al., 2010). The fat content of milk could at least partially govern the size of milk fat globules since each secreted fat globule must be enveloped by the apical plasma membrane of epithelial cells. The low fat content in camel milk (Table 1) could favor the secretion and stabilization of small fat globules.

The zeta potential values of camel and bovine milk fat globules were not significantly different (Fig. 1-D). The zeta potential values found in this study for bovine milk fat globules were in agreement with the literature (Ménard et al., 2010). These zeta potential values were significantly more negative than those recorded for human milk in the same buffer, i.e., ζ<sub>HMFG</sub> = -6.3 ± 0.2 mV (Lopez & Ménard, 2011). This difference in zeta potential values could be attributed to differences in the MFGM composition between these milk fat globules and/or to differences in the mineral composition of the aqueous phases.

## 3.2. Centrifugal separation of camel milk: effect on fat content in the fractions, milk fat globule size distribution and zeta potential

Camel milks were submitted to centrifugal separation using a cream separator, as performed in the dairy industry. The fat contents and calculated mean diameters of fat globules determined in the independent triplicates are presented in Table 1. Considering all the technological experiments performed, centrifugal separation produced camel creams with a fat content of 317.8 ± 12.1 g/kg (1.1 ± 0.1 kg of cream from 20 kg of camel milk). The fat content in the aqueous fractions was 16.7 ± 1.4 g/kg (Fig. 2A), which corresponded to partially skim milk. An amount of 18.9 ± 0.1 kg of aqueous fraction was recovered from the 20 kg of camel milk used for the centrifugal separation. The calculated fat balance revealed that the aqueous fraction obtained from the centrifugal separation contained 46.5 ± 2.1 g per 100g of the camel fat initially present in the whole camel milk. Interestingly, the aqueous fraction could be considered as a semi-skim milk for a direct commercial valorization. Our results are in agreement with a recent work reported in

the literature (Kashaninejad & Razavi, 2020), which aimed at preparing cream from camel milk (milk fat content of 31 g/kg), and the skim milk obtained showed a fat content of 16 g/kg using a pilot plant cream separator.

To perform a comparative analysis between camel milk and bovine milk, the same cream separator was used with bovine milk to recover bovine cream and the respective aqueous fraction. Bovine creams with a fat content of 395.4 ± 10.3 g/kg (2.0 ± 0.1 kg of cream from 20 kg of bovine milk) were obtained. The aqueous fraction contained 1.2 ± 0.1 g/kg fat, which corresponds to skim milk (production of 18.1 ± 0.1 kg of skim milk from 20 kg of bovine milk) (Fig. 2A). The aqueous fraction recovered from the camel milk contained 14-fold more fat content than the aqueous fraction recovered from bovine milk (16.7 ± 1.4 g/kg vs. 1.2 ± 0.1 g/kg, respectively). The fat content in the aqueous fraction corresponded to 2.8 ± 0.1 g per 100 g of total fat for bovine milk vs. 46.5 ± 2.1 g per 100 g of total fat for camel milk. The smaller amount of fat lost in the bovine milk aqueous fraction corresponds to a greater efficiency of fat globule recovery upon centrifugal separation of bovine milk compared to camel milk.

Representative size distributions of camel milk fat globules in the whole milk and fractions obtained by centrifugal separation, i.e., the creams and aqueous fraction, are presented in Fig. 2-B. The characteristic parameters calculated from the size distributions are presented in Table 1. The large milk fat globules were concentrated in the cream, while the small milk fat globules were recovered in the aqueous fraction. In the cream, the size distribution of milk fat globules ranged from 1 to 6 µm, with a mean diameter of 3.2–3.5 ± 0.2 µm. In the aqueous fraction, the size of milk fat globules ranged from 35 nm to 1.4 µm, with two main populations centered at 0.12 µm and 0.6 µm, and a mean diameter $d_{43}$ = 0.43–0.54 ± 0.2 µm. For comparison, Fig. 2-B shows the size distribution of camel milk fat globules in a cream prepared using an industrial-scale cream separator in Mauritania by the dairy company, Tiviski Camel Milk Dairy (Nouakchott). The size distribution of milk fat globules in this industrial cream ranged from 1 to 8 µm, with a mean
<caption>**Table 1**</caption>

Fat content, size parameters and apparent zeta potential of fat globules in camel whole milk, creams and aqueous fractions obtained by disc stack centrifugal separation from the corresponding whole milks (means ± standard deviation; n = 3) using a centrifugal separator at pilot plant scale. The centrifugal separation was performed from three independent whole camel milks, denoted #1, #2 and #3.

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Fat content<br>(g/kg)</th><th colspan="3">Size parameters</th><th rowspan="2">Zeta-<br>potential<br>(mV)</th></tr><tr><th>Mode<br>(µm)</th><th>D<sub>43</sub><br>(µm)</th><th>Surface<br>(m<sup>2</sup>/g fat)</th></tr></thead><tbody><tr><td>Whole milk<br>#1</td><td>31.2<sup>a</sup> ± 0.2</td><td>2.6<sup>ab</sup> ±<br>0.2</td><td>2.2<sup>a</sup> ±<br>0.1</td><td>13.6<sup>a</sup> ± 0.2</td><td>-11.8 ± 0.5</td></tr><tr><td>Cream #1</td><td>305.2<sup>b</sup><br>±10.2</td><td>2.9<sup>ab</sup><br>±0.2</td><td>3.2<sup>b</sup><br>±0.1</td><td>2.3<sup>b</sup><br>±0.1</td><td>-11.6 ± 0.8</td></tr><tr><td>Aqueous<br>fraction<br>#1</td><td>15.1<sup>c</sup><br>±0.2</td><td>0.6<sup>c</sup><br>±0.1</td><td>0.43<sup>c</sup><br>±0.02</td><td>36.1<sup>c</sup><br>±0.3</td><td>-12.1 ± 0.8</td></tr><tr><td><sup>a</sup>Stats size<br>#1</td><td>P < 0.001</td><td>P <<br>0.01</td><td>P <<br>0.001</td><td>P < 0.001</td><td>NS</td></tr><tr><td>Whole milk<br>#2</td><td>33.7<sup>a</sup> ± 0.2</td><td>2.5<sup>ab</sup> ±<br>0.3</td><td>2.4<sup>a</sup> ±<br>0.2</td><td>14.2<sup>a</sup> ± 0.1</td><td>-11.7 ± 0.8</td></tr><tr><td>Cream #2</td><td>319.5<sup>b</sup> ±<br>6.2</td><td>2.8<sup>ab</sup> ±<br>0.2</td><td>3.4<sup>b</sup> ±<br>0.2</td><td>2.6<sup>b</sup> ± 0.1</td><td>-12.1 ± 0.7</td></tr><tr><td>Aqueous<br>fraction #2</td><td>17.2<sup>c</sup><br>±0.2</td><td>0.6<sup>c</sup><br>±0.1</td><td>0.48<sup>c</sup><br>±0.02</td><td>33.2<sup>c</sup><br>±0.2</td><td>-11.8 ± 0.5</td></tr><tr><td><sup>a</sup>Stats size<br>#2</td><td>P < 0.001</td><td>P <<br>0.01</td><td>P <<br>0.001</td><td>P < 0.001</td><td>NS</td></tr><tr><td>Whole milk<br>#3</td><td>33.2<sup>a</sup> ± 0.2</td><td>2.6<sup>a</sup> ±<br>0.2</td><td>2.6<sup>a</sup> ±<br>0.1</td><td>13.8<sup>a</sup> ± 0.1</td><td>-11.8 ± 0.6</td></tr><tr><td>Cream #3</td><td>328.7<sup>b</sup> ±<br>5.2</td><td>3.1<sup>b</sup> ±<br>0.2</td><td>3.5<sup>b</sup> ±<br>0.2</td><td>2.8<sup>b</sup> ± 0.1</td><td>-12.3 ± 0.7</td></tr><tr><td>Aqueous<br>fraction<br>#3</td><td>17.8<sup>c</sup> ±<br>0.2</td><td>0.6<sup>c</sup><br>±0.1</td><td>0.54<sup>c</sup><br>±0.02</td><td>34.3<sup>c</sup><br>±0.3</td><td>-11.6 ± 0.5</td></tr><tr><td><sup>a</sup>Stats size<br>#3</td><td>P < 0.001</td><td>P <<br>0.001</td><td>P <<br>0.001</td><td>P < 0.001</td><td>NS</td></tr></tbody></table>

NS = non-significant; P > 0.05.

a P-values calculated by ANOVA.

diameter of 3.6 ± 0.2 µm. The presence of larger fat globules in the industrial camel cream compared to the pilot-scale camel cream prepared in our lab was interpreted as coalescence, i.e., the physical destabilization of part of the milk fat globules.

The apparent zeta potential values of camel milk fat globules recovered in the creams and the aqueous fractions were not significantly different (Table 1). This indicated that the zeta potential values were similar regardless of the size of camel milk fat globules, and that the fractionation of milk fat globules performed in the centrifugal separator did not affect the surface properties of milk fat globules. This was interpreted as an absence of process-induced alteration of the camel MFGM under centrifugal force.

The greater recovery of fat obtained with bovine milk associated with a smaller amount of fat in the aqueous fraction compared to camel milk was due to the large size of bovine milk fat globules (high proportion of fat globules above 1 µm). This result highlights the fact that it is important to adapt dairy processing to the specific characteristics of the milk used, particularly to the size of milk fat globules, to avoid loss of fat during cream preparation and/or to develop the valorization of the aqueous fraction recovered after centrifugal separation. The semi-skim milk (fat content 16.7 g/kg) recovered from camel milk that contained small camel milk fat globules (size below 1 µm) covered by the MFGM could be valorized for its high-nutritional value. The size of milk fat globules plays a key role in the digestive tract by providing an interfacial area where the digestive enzymes can adsorb. It has been reported that the small size of milk fat globules covered by their biological membrane increases their hydrolysis by digestive enzymes (Berton et al., 2009, 2012; Garcia et al., 2014) and affects the metabolic behavior of dietary milk lipids (Michalski et al., 2006). The small size of milk fat globules in camel milk could therefore promote their efficient hydrolysis in the

gastrointestinal tract during digestion. Further studies dedicated to the digestibility of camel milk fat globules in whole milks, creams and semi-skim milk fractions should be conducted *in vitro* and *in vivo* in clinical studies involving infants and adults.

### 3.3. Lipid composition of camel milk fat globules as a function of their size

#### 3.3.1. Fatty acids

The fatty acid composition of camel milk reported in this study (Fig. 3) was in agreement with previous reports (Attia et al., 2000; Berhe et al., 2017; Walter et al., 2020). As expected, camel milk fat globules contained a very small proportion of short-chain fatty acids (from C4:0 to C12:0). The saturated fatty acids accounted for 56.3 g/100 g fatty acids, mainly C16:0 (~30 g/100 g fatty acids), C18:0 (~12.5 g/100 g fatty acids) and C14:0 (~11 g/100 g fatty acids). The unsaturated fatty acids accounted for 43.7 g/100 g fatty acids. The main unsaturated fatty acids were C18:1 n-9 (28.5 g/100 g fatty acids) and C16:1 n-9 (10.7 g/100 g fatty acids). The essential polyunsaturated fatty acids (PUFAs) were as follows: C18:2 n-6 (3.2 g/100 g fatty acids, linoleic acid) and C18:3 n-3 (0.3 g/100 g fatty acids, α-linolenic acid).

Fig. 3 shows the comparative analysis of the fatty acid compositions of whole camel milk with the fractions obtained by centrifugal separation, i.e., the cream fraction containing large camel milk fat globules (larger than 1 µm) and the aqueous fraction containing small camel milk fat globules (less than 1 µm; semi-skim milk). Differences in fatty acid composition of milk fat globules were determined, based on their size, from each fractionated camel milk. This was interpreted as differences in pathways of fatty acid biosynthesis between small and large camel milk fat globules. Indeed, fatty acid composition results from *de novo* synthesis in the mammary gland (chain length of C4 to C16) or is derived from the blood as preformed fatty acids after hydrolysis of triacylglycerols from the diet or adipose tissue chain length of C16 and higher (Palmquist, 2006). A proportion of fatty acids from the polar lipids located in the biological membrane surrounding the fat globule could also contribute to differences in the fatty acid composition. The small milk fat globules (size less than 1 µm) recovered from semi-skim milk contained significantly lower amounts of saturated fatty acids than large milk fat globules (larger than 1 µm): 54.1 vs. 55.8 g/100 g fatty acids, respectively. They also contained a significantly higher amount of PUFAs: 5.7 vs. 3.5 g/100 g fatty acids, respectively. Regarding the individual fatty acids, the small milk fat globules contained significantly larger amounts of C16:1 n-9 and C18:2 n-6 than large milk fat globules concentrated in the cream and the whole milk. They also contained significantly smaller amounts of C16:0, C18:0 and C18:1 n-9 than large milk fat globules and the whole milk (Fig. 3).

Differences in fatty acid composition between whole bovine milks producing small or large milk fat globules, and between small and large milk fat globules fractionated from the same milk have been previously reported in the scientific literature. Using centrifugal separation, Timmen and Patton (1988) reported differences in the fatty acid compositions of bovine milk fat globules depending on their size. They determined that the small fat globules (1–1.5 µm) remaining in the skim milk fraction contained less C4:0 to C10:0 and C18:0 and more C18:1 n-9 than did the large fat globules concentrated in the cream (about 3 µm average diameter). They interpreted these differences as a necessity for the liquidity of milk fat globules during their biosynthesis. The smaller amount of C18:0 in small camel milk fat globules is in agreement with previous works reporting significantly (P < 0.0001) less C18:0 in small bovine milk fat globules than in large ones fractionated by microfiltration (Lopez et al., 2011).

#### 3.3.2. Cholesterol

The amount of cholesterol was found to be higher in camel milk than in bovine milk: 3.6–3.7 ± 0.2 vs. 3.3 ± 0.2 mg/g fat, respectively (Table 2), in agreement with the literature (Al haj & Al Kanhal, 2010). This could be attributed at least partially to the small size of camel milk
<figure><img src="image_4.png" /><figcaption>(B) Size distributions of milk fat globules (MFG)</figcaption></figure>

<figure><img src="image_5.png" /></figure>

**Fig. 2.** Fractionation of camel milks carried out by disc stack centrifugal separation using a conventional cream separator. (A) Fat contents in the whole milks, creams and aqueous fractions: comparison between camel milks and bovine milks; (B) Representative size distributions of camel milk fat globules in the whole milk, cream and aqueous fraction recovered after centrifugal separation, comparison with an industrial camel cream.

<figure><img src="image_6.png" /></figure>

**Fig. 3.** Fatty acid compositions of the milk fat globules (MFG) from whole camel milks, creams and aqueous fractions recovered after disc stack centrifugal separation that induced fractionation as a function of the size of MFG. The creams contained large (greater than 1 µm) camel MFG, whereas the aqueous fractions corresponding to semi-skim milk contained small (less than 1 µm) camel MFG. Results correspond to means and standard deviation (n = 9); (***), P < 0.01; **, P-values calculated by ANOVA.

fat globules compared to bovine milk and then to the greater amount of MFGM where cholesterol is located within the polar lipids. The amount of cholesterol (expressed in mg/g fat) in the industrial cream produced in Mauritania was similar to the amount found in the camel milks from Mauritania and Tunisia (Table 2). The small milk fat globules contained in the semi-skim milk fraction contained a greater amount of cholesterol than the large fat globules concentrated in the cream: 3.8 ± 0.2 vs. 3.5 ± 0.2 mg/g fat (Table 2). This was interpreted as the greater amount of MFGM surrounding small milk fat globules than large ones.

### 3.3.3. Polar lipids

Total polar lipid (PL) concentrations in camel and bovine milks were calculated as the sum of glycerophospholipids (PE, PI, PS, and PC) and SM concentration (Table 2). The amounts of PL were similar in milks from Mauritania and Tunisia: 17.9–18.2 ± 0.6 mg PL per g of fat. This was consistent with similar milk fat globule size distributions (Fig. 1).

The whole milks, creams and aqueous fractions recovered by centrifugal separation contained significantly different amounts of polar lipids, expressed per unit g of fat for comparison. The whole milks contained 18.2 ± 0.5 mg PL per g of fat, whereas the creams contained 13.6 ± 0.4 PL per g of fat and the aqueous fractions contained 37.4 ± 2.4 PL per g of fat. These differences in polar lipid contents were linked to differences in the diameter of fat globules and the surface area of the MFGM around the fat globules (Fig. 2B). These results showed that the aqueous fraction, i.e., the semi-skim milk fraction, recovered as a co-product of camel cream preparation is a highly interesting source of milk polar lipids and MFGMs around small milk fat globules.

The relative proportion of individual polar lipids was similar between milks from Mauritania and Tunisia, and was attributed to camel milk regardless of the geographical origin. The main polar lipid was SM, accounting for about 40 g per 100 g of total PL. Differences were observed depending on the size of camel milk fat globules, with a significantly higher proportion of PC and a lower proportion of PE in large milk fat globules concentrated in the creams compared to small globules in the semi-skim milk fraction (Table 2).

The amount of polar lipids in camel milk is 5-fold higher than the amount of polar lipids determined for bovine milks (17.9–18.2 ± 0.5 vs. 3.6 ± 0.5 mg polar lipids per g of fat, respectively; Table 2). The amount of polar lipids in camel milk was greater than those determined for milk of other species: 10.5 ± 0.2 mg PL/g fat for goat milk (Et-Thakafy et al., 2017); 7.0 ± 0.1 mg PL/g fat for sheep milk (Et-Thakafy et al., 2017); 2.5–8.8 mg PL/g fat for bovine milk (Et-Thakafy et al., 2017; Luo et al., 2018); 3.1–4.1 ± 0.2 mg PL/g fat for human milk (Lopez & Ménard,
<caption>**Table 2**</caption>

Lipid composition of camel milk fat globules (MFG) as a function of their size and comparison with bovine milk. Fractions of large and small MFG were obtained by disc stack centrifugal separation from the corresponding whole milks. The centrifugal separation was performed from three independent whole camel milks. The industrial cream (noted #C) was produced by an industrial dairy. The lipid composition of whole milks obtained from Tunisia (noted #T) was added for comparison with the milks from Mauritania used for the centrifugal fractionation.

<table><thead><tr><th rowspan="3">Membrane lipids</th><th colspan="2">Camel milk</th><th rowspan="3"><sup>a</sup>Stats<br/>#size</th><th rowspan="3">Industrial<br/>Cream (#C)</th><th rowspan="3">Whole milk (#T)</th><th rowspan="3">Whole bovine milk</th><th rowspan="3"><sup>a</sup>Stats<br/>#milk<br/>DvsB</th></tr><tr><th rowspan="2">Whole milk</th><th colspan="2">Fractions</th></tr><tr><th>Large MFG</th><th>Small MFG</th></tr></thead><tbody><tr><td>Cholesterol (mg/g fat)</td><td>3.6<br/>±0.2</td><td>3.5<br/>±0.2</td><td>3.8<br/>±0.2</td><td>***</td><td>3.5<br/>±0.2</td><td>3.7<br/>±0.2</td><td>3.3<br/>±0.2</td><td>***</td></tr><tr><td>Polar lipids (mg/g fat)</td><td>18.2<br/>±0.5</td><td>13.6<br/>±0.4</td><td>37.4<br/>±2.4</td><td>***</td><td>15.2<br/>±1.2</td><td>17.9<br/>±0.6</td><td>3.6<br/>±0.5</td><td>***</td></tr><tr><td colspan="9">Relative proportion of individual polar lipids (%; mg PL per 100 mg PL)</td></tr><tr><td>PE</td><td>18.3<br/>±0.7</td><td>16.8<br/>±0.8</td><td>20.7<br/>±0.6</td><td>***</td><td>18.5<br/>±0.2</td><td>19.1<br/>±0.9</td><td>28.7<br/>±1.1</td><td>***</td></tr><tr><td>PI</td><td>9.1<br/>±0.8</td><td>9.3<br/>±0.8</td><td>9.1<br/>±0.6</td><td>NS</td><td>9.0<br/>±0.2</td><td>9.1<br/>±1.2</td><td>10.8<br/>±1.2</td><td>NS</td></tr><tr><td>PS</td><td>7.4<br/>±0.3</td><td>7.2<br/>±0.4</td><td>7.3<br/>±0.3</td><td>NS</td><td>7.4<br/>±0.3</td><td>7.3<br/>±0.2</td><td>13.8<br/>±0.9</td><td>***</td></tr><tr><td>PC</td><td>24.4<br/>±2.1</td><td>26.1<br/>±2.6</td><td>22.1<br/>±1.8</td><td>***</td><td>26.6<br/>±1.4</td><td>23.3<br/>±3.2</td><td>22.6<br/>±2.8</td><td>NS</td></tr><tr><td>SM</td><td>40.8<br/>±3.6</td><td>40.6<br/>±2.3</td><td>40.8<br/>±1.8</td><td>NS</td><td>38.5<br/>±1.2</td><td>41.2<br/>±5.3</td><td>24.3<br/>±1.3</td><td>***</td></tr><tr><td colspan="9">Individual polar lipids (mg PL per g of fat)</td></tr><tr><td>PE</td><td>3.33<br/>±0.18</td><td>2.8<br/>±0.12</td><td>7.74<br/>±0.62</td><td>***</td><td>2.83<br/>±0.15</td><td>3.40<br/>±0.26</td><td>1.03<br/>±0.18</td><td>***</td></tr><tr><td>PI</td><td>1.66<br/>±0.12</td><td>1.26<br/>±0.08</td><td>3.40<br/>±0.21</td><td>***</td><td>1.36<br/>±0.11</td><td>1.63<br/>±0.25</td><td>0.39<br/>±0.08</td><td>***</td></tr><tr><td>PS</td><td>1.35<br/>±0.12</td><td>0.98<br/>±0.07</td><td>2.73<br/>±0.18</td><td>***</td><td>1.11<br/>±0.18</td><td>1.31<br/>±0.08</td><td>0.49<br/>±0.07</td><td>***</td></tr><tr><td>PC</td><td>4.44<br/>±0.56</td><td>3.55<br/>±0.27</td><td>8.27<br/>±0.73</td><td>***</td><td>4.06<br/>±0.62</td><td>4.17<br/>±0.69</td><td>0.81<br/>±0.11</td><td>***</td></tr><tr><td>SM</td><td>7.43<br/>±0.66</td><td>5.52<br/>±0.42</td><td>15.26<br/>±0.84</td><td>***</td><td>5.83<br/>±0.43</td><td>7.34<br/>±0.77</td><td>0.87<br/>±0.12</td><td>***</td></tr></tbody></table>

(#C): camel milk cream provided by a dairy plant in Nouakchott (Mauritania); fat content: 42 %wt.

(#T): camel milk collected in Tunisia in June; n = 3 milks.

Abbreviations: PL = polar lipids; PE = phosphatidylethanolamine; PI = phosphatidylinositol; PS = phosphatidylserine; PC = phosphatidylcholine; SM = sphingomyelin.

a Results are means ± standard deviation (n = 6).

a P-values calculated by ANOVA. (*** ) P < 0.01.

2011); 2.6 ± 0.5 mg PL/g fat for buffalo milk (Ménard et al., 2010); and 5.1 ± 0.2 mg PL/g fat for yak milk (Luo et al., 2018). The large amount of polar lipids per g of fat characterized in camel milk was attributed to the small size of camel milk fat globules and then to the large surface area covered by the camel MFGM, as previously discussed (Lopez et al., 2011; Ménard et al., 2010).

Regarding the relative proportions of the individual milk polar lipids, the results showed that zwitterionic polar lipids PE, PC and SM were quantitatively the major polar lipids, whereas the anionic polar lipids PI and PS were relatively minor regardless of milk sample species. These results were in accordance with previous reports (Et-Thakafy et al., 2017; Lopez et al., 2011; Lopez & Ménard, 2011; Luo et al., 2018; Ménard et al., 2010). Camel MFGM contained a significantly higher proportion of SM than bovine MFGM: 41.2 ± 5.3 g/100 g PL vs. 24.3 ± 1.3 g/100 g PL, respectively. Interestingly, this high proportion of SM in camel milk is close to values reported in human milk, i.e., 32–46 g/100 g PL in human breast milk (Lopez & Ménard, 2011). The proportion of SM in camel milk was higher than the amount reported for milk of other species, i.e., 33 g/100 g PL in yak milk (Luo et al., 2018), 28 g/100 g PL in sheep milk (Et-Thakafy et al., 2017), 22 g/100 g PL in goat milk (Et-Thakafy et al., 2017), 25 g/100 g PL in buffalo milk (Ménard et al., 2010), and 24–27 g/100 g PL in bovine milk (Lopez et al., 2014; Luo et al., 2018; Ménard et al., 2010). Camel milk contained 7.34 mg SM per g of fat and 228 mg SM per kg of milk, whereas bovine milk contained 0.87 mg SM per g of fat and 34 mg SM per kg of milk (calculations performed from values reported in Table 2). Camel milk is therefore an important source of SM in the human diet where populations consume

this milk.

The high proportion of SM among camel milk polar lipids is particularly interesting. SM is the main sphingolipid naturally present in milk, and whose nutritional importance in the human diet has been reported for two decades (Vesper et al., 1999). Dietary SM was reported to play an important role as a precursor to several bioactive metabolites (ceramide, sphingosine and sphingosine phosphate) with considerable activity in the gut, and has been associated with many health benefits (Duan & Nilsson, 2009). Scientific knowledge of the role of sphingolipids in infant gut health and immunity has been recently reviewed (Nilsson, 2016). Milk-SM would play an important role in neonatal gut maturation during the suckling period. For example, milk-SM was reported to accelerate enzymatic and morphological maturation of the intestine in artificially reared rats (Motouri et al., 2003). Milk-SM has also been reported to lower cholesterol absorption in adults and subsequently have a hypocholesterolemic effect (Noh & Koo, 2004; Nyberg et al., 2000; Al haj & Al Kanhal, 2010). This could at least be partially due to the attractive interactions between milk-SM and cholesterol leading to the formation of complexes in the gastro-intestinal tract (Cheng et al., 2017; Murthy et al., 2015) and their poor solubilization by the bile salts during digestion (Tai et al., 2021). According to Nilsson (2016), it seems unlikely that the milk-SM in the MFGM would markedly inhibit cholesterol absorption in suckling neonates. The large amount of SM in camel milk could be of physiological and nutritional interest and could positively contribute to the prevention of coronary heart disease, which is one of the main causes of death in industrialized countries.
### 3.4. Microstructure of the camel MFGM regardless of the size of milk fat globules

The microstructure of the biological membrane surrounding camel milk fat globules was observed in *situ* in the milks, creams and semi-skim milks using confocal microscopy and pertinent fluorescent dyes able to specifically stain the glycosylated components (i.e., glycoproteins and glycolipids) and the fluid matrix of polar lipids. The small size of fat globules in camel milks (i.e., 2.2 µm, Fig. 1), and particularly in the semi-skim milks (i.e., 0.5 µm, Table 1) made it difficult to examine the microstructure of their biological membrane due to the resolution of the CLSM technique. However, to the author's knowledge, this is the first time that a heterogeneous distribution of glycoproteins and polar lipids was observed at the surface of camel milk fat globules regardless of their size.

CLSM images presented in Fig. 4 were obtained with the double labeling of the glycosylated compounds and fluid polar lipids. The presence of glycosylated compounds surrounding the MFGM of each camel milk fat globule regardless of their size was observed (Fig. 4-A). The main glycosylated compounds corresponded to the MFGM glycoproteins. The glycoproteins were heterogeneously distributed in the MFGM and protruded into the aqueous phase forming a glycocalyx around the camel milk fat globules. The glycoproteins were mainly distributed as patches of various sizes (Fig. 4-B – 4-E). Such a heterogeneous distribution of MFGM-glycoproteins was in agreement with previous studies focused on milk of other mammal species (Lopez et al., 2010; Lopez &

Ménard, 2011; Luo et al., 2018; Nguyen et al., 2015). Non-fluorescent areas, mainly circular in shape (Fig. 4-B, 4-C and 4-D), were observed in camel MFGM. These areas did not contain any glycosylated compounds or fluid polar lipids. They were attributed to a heterogeneous distribution of polar lipids in the camel MFGM. CLSM observations performed at different Z-depths allowed the characterization of the surface of fat globules (denoted Z in Fig. 4), and of their equatorial section (denoted Z + dz in Fig. 4) showing the non-labeled core of fat globules composed of triacylglycerols (TAG) and the surrounding MFGM (Fig. 4-F and 4-G). The decomposition of the CLSM images allowed the observation of the spatial distribution of the WGA fluorescent dye only and showed the heterogeneous distribution of the glycoproteins at the surface of milk fat globules (green color, Fig. 4-F and 4-G). The heterogeneous distributions of glycoproteins and polar lipids in the MFGM have been observed regardless of the size of camel milk fat globules, in agreement with previous reports on whole, small and large bovine milk fat globules (Lopez et al., 2011).

The heterogeneous distribution of polar lipids in the camel MFGM was investigated by CLSM with exogenous head-labeled phopholipid Rh-DOPE as a fluorescent dye (Fig. 5). Fig. 5-A and 5-B show the fluorescent labeling of polar lipids in the MFGM surrounding camel and bovine milk fat globules, respectively. CLSM observations of bovine MFGM performed in this study were in agreement with the pioneer work performed by our group (Lopez et al., 2010). CLSM images showed that camel milk fat globules had a heterogeneous distribution of polar lipids on their surface (Fig. 5-A). CLSM observations also showed the presence

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Confocal laser scanning microscopy (CLSM) images of milk fat globules in camel milks with the fluorescent labeling of the fluid matrix of phospholipids (red color; rhodamine-DOPE fluorescent dye) and glycoproteins (yellow color with the double labeling or green color; WGA fluorescent dye) in the milk fat globule membrane. The presence of non-fluorescent areas corresponding to sphingomyelin (SM)-rich domains is highlighted by arrows. Abbreviation: TAG = triacylglycerols.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Confocal laser scanning microscopy (CLSM) images of milk fat globules in (A) camel milk and (B) bovine milk, with the labeling of the fluid matrix of phospholipids in the milk fat globule membrane (red color; fluorescent dye = rhodamine-DOPE). The non-fluorescent areas correspond either to the core of milk fat globules rich in triacylglycerols (TAG) or to sphingomyelin (SM)-rich domains in the milk fat globule membrane when the surface of milk fat globules is observed. The SM-rich domains are highlighted by white arrows. The presence of a cytoplasmic remnant is also highlighted.</figcaption></figure>

of cytoplamic remnants around fat globules (Fig. 5-A3), as previously reported with similar methodological conditions (Lopez & Ménard, 2011; Nguyen et al., 2015). The non-fluorescent (black) areas observed in the camel MFGM were interpreted as the lateral segregation of polar lipids between the fluid matrix into which the fluorescent dye Rh-DOPE was able to integrate and the ordered lipid domains where the fluorescent dye was excluded (Fig. 5). Non-fluorescent areas in the camel MFGM were interpreted as the formation of ordered SM-rich lipid domains, in agreement with pioneering CLSM observations of MFGM in bovine milk (Lopez et al., 2010). This interpretation is based on previous studies revealing the main structural role played by SM in the architecture of the MFGM, mainly the lateral phase separation of SM domains in an ordered state from the fluid matrix composed of unsaturated polar lipids (Lopez et al., 2019). Among milk polar lipids, SM molecules are able to form ordered domains due to their specific composition that is

rich in very long acyl chains that change their physical state as a function of temperature, with a melting phase transition temperature $T_m$ = 34.3 °C (Lopez et al., 2019).

Cholesterol molecules were not directly observed in the MFGM using CLSM. However, cholesterol is a main compound of the camel MFGM (Table 2). Moreover, it has been previously reported that cholesterol can solubilize in milk SM bilayers and lead to the formation of liquid-ordered phase complexes through attractive interactions (Cheng et al., 2017; Lopez et al., 2018). The liquid-ordered phase domains formed by cholesterol and milk SM are formed in the MFGM and remain present regardless of the temperature, as previously reported in bovine, human and buffalo MFGMs (Et-Thakafy et al., 2017; Lopez & Ménard, 2011; Nguyen et al., 2016). The number of ordered lipid domains ranged from zero to more than ten on one side of the camel milk fat globules. Most of the lipid domains observed in the camel MFGM were circular in shape, in
agreement with previous observations performed after storage below T_m (Et-Thakafy et al., 2017). The circular micro-domains observed in the camel MFGM were in agreement with previous observations in the bovine MFGM after stabilization at room temperature (e.g., 19 ± 1 °C) or refrigerated temperatures, i.e., below T_m (Et-Thakafy et al., 2017; Lopez et al., 2010, 2011).

The large amount of SM in camel milk, i.e., 41.2 ± 5.3 g/100g of total PL in the MFGM (Table 2) correlated well with the large surface area of the outer bilayer of the camel MFGM exhibiting SM-rich domains. These circular microdomains have been reported to induce topographical heterogeneities in the plane of the outer bilayer of the MFGM as characterized in model systems, with monolayers of milk polar lipids in dry conditions (Lopez, 2020b) and bilayers of milk polar lipids in hydrated conditions (Lopez et al., 2019). These microdomains also induce mechanical heterogeneities in the membrane since they are more rigid than the surrounding fluid phase composed of unsaturated polar lipids (Lopez et al., 2019). The phase separation between ordered lipids segregated in domains, and the fluid phase of polar lipids in which the anionic phospholipids PS and PI are dispersed, governs the preferential interaction with proteins, as previously demonstrated (Obeid et al., 2019). The presence of circular microdomains enriched in SM and

cholesterol in an ordered phase on the surface of camel milk fat globules could be involved in the mechanism of milk fat globule digestion by digestive enzymes (Berton et al., 2012; Tai et al., 2021).

Fig. 6 proposes a schematic representation of camel milk fat globules covered with their biological membrane in which polar lipids and proteins exhibit a heterogeneous distribution. The lipid composition of the camel milk fat globule membrane characterized by a high proportion of SM and cholesterol govern its structure and probably its functions. The structure of the outer bilayer of the camel MFGM results from the lateral phase separation of camel milk polar lipids with the formation of ordered lipid domains composed of SM and cholesterol, which are surrounded by the fluid matrix of polar lipids composed of unsaturated polar lipids. These ordered domains are generally micron-sized and circular, as observed in this study by confocal microscopy. The membrane proteins appeared to be excluded from ordered lipid domains and preferentially located in the fluid matrix of polar lipids.

## 4. Conclusion

Camel milk contains a high proportion of milk fat globules smaller than 1 µm, which can be lost during the processing of milk into dairy

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Schematic representations of the microstructure of camel milk fat globules and their biological membrane (not to scale) showing the heterogeneous distribution of proteins and polar lipids. The polar lipid composition in the camel milk fat globule membrane is dominated by sphingomyelin molecules, which play a key structural role. The lateral phase separation of polar lipids leads to the formation of ordered lipid domains composed of sphingomyelin and cholesterol surrounded by the fluid matrix composed of the unsaturated polar lipids. Abbreviations: MFGM = milk fat globule membrane; TAG = triacylglycerols; SM = sphingomyelin; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PI = phosphatidylinositol, PS = phosphatidylserine.</figcaption></figure>
products (e.g., cream, butter). This study showed that a size-based
fractionation of camel milk fat globules occurs during the preparation
of creams using conventional centrifugal separators. Also, for the first
time to the author's knowledge, this study revealed that the high con-
centration of fat recovered in the aqueous fraction, i.e. 16 g/kg, could be
valorized as semi-skim milk to avoid losses of fat in the co-product of
cream preparation. The lipid composition of camel milk fat globules was
determined and small differences were observed depending on their
size. The main difference is the amount of polar lipids from the MFGM,
which is higher per g of fat for the small fat globules. Camel milk con-
tains a large amount of polar lipids in the MFGM compared to milk of
other species, e.g., bovine milk. Moreover, the relative proportion of
sphingomyelin (SM) is higher in the camel MFGM than that determined
for the other milks. Camel milk is therefore an important source of di-
etary SM for human nutrition. Observations of camel MFGM micro-
structure revealed a heterogeneous distribution of glycoproteins and
polar lipids with the formation of ordered SM-rich domains. These
microdomains were circular in shape and devoid of proteins. This study
has increased scientific knowledge about camel milk fat globules and
their biological membrane. The specific composition and structural
characteristics of camel milk fat globules, especially their high amount
of SM in the MFGM, may contribute to their efficient digestion and
nutritional properties. These results open perspectives for the valoriza-
tion of the aqueous fraction of camel milk rich in small milk fat globules
that is recovered as a co-product of camel cream preparation performed
using conventional cream separators.

CRediT authorship contribution statement

**Imène Felfoul:** Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Christelle Lopez:** Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

The authors warmly thank Dr. Frédéric Gaucheron for initiating in-
ternational scientific partnerships between INRAE (France), the Uni-
versity of Nouakchott Al Asriya (Mauritania) and the LAVASA-ENIS
University of Sfax (Tunisia). The dairy plant, Tiviski Camel Milk Dairy
(Nouakchott, Mauritania), is acknowledged for providing camel cream
and milk during this fruitful international partnership. The authors
thank the dairy pilot plant of INRAE STLO (Rennes, France) for tech-
nological experiments of milk fractionation. The authors are grateful to
Marielle Blot for technical support in polar lipid and fatty acid analysis.
This project was financially supported by the "PHC-Utique" program of
the French Ministry of Foreign Affairs and French Ministry of Higher
Education and Research and the Tunisian Ministry of Higher Education
and Scientific Research in the CMCU project, number 24G0903.

Data availability

Data will be made available on request.

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