Chemical and structural characterisation of almond oil bodies and bovine milk fat globules

Food Chemistry 132 (2012) 1996–2006

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Chemical and structural characterisation of almond oil bodies and bovine milk fat globules Sophie Gallier a,⇑, Keith C. Gordon b, Harjinder Singh a a b

Riddet Institute, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand MacDiarmid Institute for Advanced Materials and Nanotechnology, Department of Chemistry, University of Otago, Dunedin 9054, New Zealand

a r t i c l e

i n f o

Article history: Received 8 August 2011 Received in revised form 28 November 2011 Accepted 14 December 2011 Available online 21 December 2011 Keywords: Almond oil body Bovine milk fat globule Membrane structure Confocal laser scanning microscopy Raman microscopy Fatty acid Sterol Phospholipid

a b s t r a c t Lipids in almonds are present as oil bodies in the nut. These oil bodies are surrounded by a membrane of proteins and phospholipids and are a delivery vehicle of energy in the form of triglycerides, similarly to the more studied bovine milk fat globule membrane. Chemical, physical and microscopic analyses revealed major differences in the composition and structure of almond oil bodies and bovine milk fat globules. The lipids of both natural emulsions differed in degree of unsaturation, chain length, and class. The almond oil body membrane does not contain any cholesterol or sphingomyelin unlike the bovine milk fat globule membrane. Therefore, the phospholipid distribution at the surface of the oil bodies did not present any liquid-ordered domains. The membranes, a monolayer around almond oil bodies and a trilayer around bovine fat globules, may affect the stability of the lipid droplets in a food matrix and the way the lipids are digested. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Almonds are considered to be a great source of proteins, dietary fibre, health-promoting unsaturated fatty acids, vitamin E, other vitamins, minerals; they are also low in saturated fats, and contain no cholesterol (Miraliakbari & Shahidi, 2008). As a result, almonds are used in several food products, such as almond-based beverages, pastes, butter, snacks and baking goods. Almond lipids contain 91.6% unsaturated fatty acids, possibly contributing to the cardioprotective effect of a diet rich in nuts (Maguire, O’Sullivan, Galvin, O’Connor, & O’Brien, 2004). Many of the health benefits of almonds are attributed to their high oleic acid content (70% of the fatty acids). Almonds may also have potential action against obesity-related diseases (Chen, Lapsley, & Blumberg, 2006). Most of the research on almond lipids has focused on their chemical composition (Maguire et al., 2004; Miraliakbari & Shahidi, 2008; Venkatachalam & Sathe, 2006). In addition to triglycerides, almond lipids contain phytosterols, phospholipids, sphingolipids and tocopherol (mainly a-tocopherol). Almond lipids, about 55% of the nut weight, are stored in oil bodies (OB) which are lipid droplets surrounded by a monolayer of phospholipids embedding proteins (Beisson et al., 2001). These pro-

⇑ Corresponding author. Tel.: +64 (0)6 356 9099x81612; fax: +64 (0)6 350 5655. E-mail address: [email protected] (S. Gallier). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.12.038

teins are mainly oleosins and have a low molecular weight. Oleosins have been shown to cover entirely the phospholipid monolayer, preventing its hydrolysis by phospholipases, and are thought to stabilise oil bodies from coalescence (Huang, 1994). Huang (1994) introduced a model of the seed OB structure; one domain of the oleosin covers the phospholipid surface, another domain penetrates the monolayer and a last domain anchors the triglyceride matrix. Thus the structure of the seed OB appears to be very different from raw bovine milk fat globules (MFG), which are surrounded by a phospholipid trilayer and a complex mixture of proteins, some of them glycosylated, and cholesterol (Walstra, Wouters, & Geurts, 2006). The almond membrane does not contain any sphingomyelin and only 0.02–0.03 mg cholesterol/g almond lipid (Miraliakbari & Shahidi, 2008). In contrast, sphingomyelin accounts for one third of the bovine phospholipids (Gallier, Gragson, Cabral, JimenezFlores, & Everett, 2010) and bovine milk contains 2–3 mg cholesterol/g lipid (Viturro, Meyer, Gissel, & Kaske, 2010). Both molecules are responsible for phase separation on the membrane surface (Gallier, Gragson, Jimenez-Flores, & Everett, 2010). However, almonds are rich in phytosterols, about 2.2–2.6 g/kg lipid (Maguire et al., 2004). Phytosterols and cholesterol play a role in fluidity and water permeability of membranes from plants and animals, respectively (Yankah, 2006). Phytosterols are poorly absorbed by the human intestine and reduce cholesterol absorption by decreasing the solubility of cholesterol in bile salt micelles during intestinal digestion (Yankah, 2006). In addition to a stabilising role,

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oleosins are possible receptors for lipase binding during germination of the seed (Huang, 1994). In bovine milk, the lateral organisation of the phospholipids (phase coexistence) and the presence of a glycocalyx (carbohydrate moieties from glycoproteins and glycolipids) at the surface of the globule membrane may affect the binding of lipase. Therefore the physicochemical properties of the membranes of the almond oil bodies and bovine milk fat globules may play a key role in interactions with human gastrointestinal enzymes. By blending almonds in a Tris–HCl buffer, Beisson et al. (2001) extracted intact oil bodies. In this study, a similar method was used to obtain an aqueous suspension containing almond OB by wet disintegration. A milky suspension of almond OB was chosen as an almond-derived product, where its structure can be easily studied for particle sizing and confocal and Raman microscopic imaging, as opposed to that in a solid nut. The almond suspension system also gives a picture of how natural lipids are stabilised in an aqueous medium. This system is also close to the state of the almond lipids after the mechanical process in the mouth, where a crude emulsion is formed prior to swallowing. Here two types of dietary lipid systems, one extracted from plant and one from an animal source, were studied and compared. 2. Materials and methods All chemicals were of analytical grade and were purchased from Sigma–Aldrich Corporation (St. Louis, MO) unless specified otherwise. 2.1. Almond processing and milk preparation Raw sliced almonds (nutritional composition as stated on the packaging: fat 55.2% including 3.6% saturated fat, protein 20.0%, carbohydrate 4.4%, origin USA) were purchased fresh from a local wholesale retailer in Palmerston North, New Zealand. Fresh raw bovine milk from pasture-fed (with grass silage) Friesian cows was collected from the Massey University No. 1 Dairy Farm (Palmerston North, New Zealand) during the summer. Almonds (250 g) were soaked overnight in 1 L of Milli-Q water (18 MO
cm, purified by treatment with a Milli-Q apparatus, Millipore Corporation, Bedford, MA) at room temperature. The mixture was then placed in a wet disintegrator (Jeffress Bros Ltd., Brisbane, Australia) for 5 min. The resulting mixture was sieved through a 150-lm powder sifter to remove residual almond pieces. The milky almond aqueous suspension was collected and is termed as ‘‘almond milk’’. If not used the same day, sodium azide (0.02% w/v, Merck, Darmstadt, Germany) was added to the almond milk and bovine milk, and the milk was kept at 4 °C for a maximum of 2 days, unless specified otherwise. 2.2. Chemical composition Raw bovine milk and almond milk were analysed for lipid, protein and solid contents and fatty acid composition. A Mojonnier extraction (AOAC 954.02) was carried out to determine the total lipid content and the Leco combustion procedure (AOAC 968.06) was followed for the protein quantification. The total solid content was determined by a direct forced air oven drying method (AOAC 990.20). The fatty acid composition was determined according to Sukhija and Palmquist (1988) using a Shimadzu GC-17A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionisation detector fitted with a Supelco-2560 capillary column 100 m  0.25 mm  0.2 lm film thickness (Supelco, Bellefonte, PA). The oven temperature was programmed to hold at 140 °C for 5 min then to increase to 240 °C at a rate of 4 °C/min and hold

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for 38 min. The injector and detector temperatures were 250 and 255 °C, respectively. Fatty acid methyl esters were prepared and then extracted before analysis by gas chromatography. The sterol composition of bovine milk and almond milk was measured by Asure Quality Ltd. (Auckland, New Zealand) using the method developed by Laakso (2005). The phospholipid profile of freeze-dried milk samples was determined by quantitative 31Pnuclear magnetic resonance (31P NMR) performed by Spectral Service (Köln, Germany) according to the method SAA-MET0002-02, using a Bruker Avance III 600 MHz NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). A phospholipid enrichment, i.e., increasing the concentration in phospholipids per gram of sample for a more efficient analysis, was performed before 31P NMR analysis by Soxhlet extraction (recovering more than 97% of lipids). Triphenyl phosphate was used as an internal standard. All analyses were run at least in duplicate on independent milk samples. 2.3. Protein composition The almond and bovine protein compositions of both milk samples were determined by reducing tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) (Schagger & Vonjagow, 1987). Small aliquots (50 lL) of samples were treated with 200 lL of tricine sample buffer (0.2 M Tris–HCl buffer pH 6.8, 40% glycerol, 2% SDS, 0.04% Coomassie Brilliant Blue R-250, b-mercaptoethanol (19:1, v:v)) and heated at 95 °C for 5 min. After cooling down to room temperature, the samples were centrifuged for 5 min at 1000g, the fat layer was discarded, and 10 lL of subnatant were loaded onto a precast Criterion 10–20% gradient Tricine gel (Bio-Rad Laboratories Pty., Auckland, New Zealand). The gel was stained for 40 min with Coomassie Brilliant Blue R-250 (0.003% (w/v) in 10% acetic acid (Merck, Poole, England) and 20% isopropanol (Merck)). The gel was destained with a solution of 10% acetic acid and 10% isopropanol and scanned using a Molecular Imager Gel Doc XR (Bio-Rad Laboratories Pty.). Precision Plus protein unstained standards (10–250 kDa) and polypeptide SDS–PAGE standards (1.4–26.6 kDa) were obtained from Bio-Rad Laboratories Pty. 2.4. Measurement of f-potential The f-potential of freshly collected or processed samples was measured by laser Doppler velocimetry and phase analysis light scattering (M3-PALS) technique using a Malvern Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK) equipped with a 4 mW helium/neon laser at a wavelength output of 633 nm. Samples were diluted 100-fold in Milli-Q water and put in an electrophoresis cell (Model DTS 1060C, Malvern Instruments Ltd.) at 25 °C. The refractive index (RI) of bovine milk fat was 1.456 with an absorbance of 0.001, the RI of almond fat was 1.466 (Moayedi, Rezaei, Moini, & Keshavarz, 2011) with the same absorbance, and the RI of the aqueous phase (water) was 1.33. The Smoluchowski approximation was chosen to calculate the f-potential from the mobility measurement according to Henry’s law. Ten readings from a freshly-diluted individual sample were collected and the measurements were run in triplicate on three independent milk samples. 2.5. Particle size distribution A Malvern Mastersizer MSE (Malvern Instruments Ltd.) was used to determine the average droplet size of milk samples. The same refractive indices and absorbance as above were used. The Sauter-average diameter, d32, and the volume-mean diameter, d43, of the samples were measured. Mean particle diameters were calculated as the average of duplicate measurements and the

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measurements were run in triplicate on three independent milk samples. 2.6. Confocal laser scanning microscopy (CLSM) The microstructure of the bovine MFGand almond OB was studied using a confocal laser scanning microscope (Leica DM6000B, Heidelberg, Germany) with a 63-mm oil immersion objective lens. Samples were diluted 10-fold in phosphate buffered saline solution (PBS, pH 7.2). Agarose (50 lL, 0.5% w/v in Milli-Q water) was used to fix the sample (25 lL) onto a concave glass slide. Nile Red (9-diethylamino-5H-benzo[a]phenoxazine-5-one, 1 mg/ mL in dimethyl sulfoxide,1:200, v/v) was used to stain the triglycerides and Fast Green FCF (disodium2-[[4-[ethyl-[(3-sulfonatophenyl) methyl]amino]phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azani umylidene]cyclohexa-2,5-dien-1-ylidene]methyl]-5-hydroxybenzenesulfonate, 1 mg/mL in Milli-Q water,1:200, v/v) the proteins. The Alexa FluorÒ 488 conjugate of wheat germ agglutinin (WGA, 1 mg/mL in 0.2 M PBS, pH 7.4, 1:20, v/v) and the Alexa FluorÒ 488 conjugate of concanavalin A (Con A, 1 mg/mL in fresh 0.1 M sodium bicarbonate buffer, pH 8.3, 1:20, v/v) were purchased from Invitrogen (Carlsbad, CA) and used for the localisation of glycoproteins and glycolipids at the surface of the droplets. The fluorescent head group-labelled phospholipid analogue Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rd-DHPE, 1 mg/mL in chloroform, 1:100, v/v, (Invitrogen)) was used to investigate the lateral distribution of the phospholipids on the surface of the lipid droplets, as reported by Gallier, Gragson, Jimenez-Flores et al. (2010). 2.7. Confocal Raman microscopy The chemical fingerprint of the bovine MFG and almond OB were determined by confocal Raman microscopy as reported in previous work (Gallier, Gordon, Jiménez-Flores, & Everett, 2011) with slight modifications. Samples were diluted 10-fold in PBS. Milk samples (25 lL) were deposited onto a flat quartz microscopic slide and agarose (50 lL) was used to fix the samples. A quartz cover slip was then applied rapidly without excessive pressure. The Raman spectral data were collected using a Senterra dispersive Raman microscope (Bruker Optics, Ettlingen, Germany). An Olympus BX confocal microscope (Olympus Europa GmbH, Hamburg, Germany) was used to observe and acquire Nomarski differential interference contrast images of the lipid droplets. OPUS version 6.5 (Bruker Optics) was used to control the microscope and collect the spectra. An Olympus 100 objective (numerical aperture 0.9) with a 50-lm confocal pinhole was used to collect the Raman signal from the centre of the lipid droplet sphere. An excitation wavelength of 532 nm from a diode laser (Sentinel, Bruker Optics), with 5 mW power before the objective, and a 1200 groove per millimetre grating were used to record the Raman spectra of milk lipid droplets. Spectral data were recorded in the region 15– 4441 cm 1 with a resolution of 9–18 cm 1. Each spectrum was the co-addition of thirty 10-s exposures and was recorded at room temperature. Each peak intensity was measured and an average of intensity from three droplets of the same size was calculated.

3. Results and discussion 3.1. Chemical composition of almond and bovine milk samples The almond milk contained 4.3 ± 0.1% protein, 10.2 ± 0.2% lipid and 22.75 ± 0.22% total solids. Fresh bovine milk contained 3.6 ± 0.0% protein, 5.5 ± 0.1% lipid and 15.55 ± 0.01% total solids.

3.1.1. Fatty acid profiles Triglycerides are the main lipids in both milk types (Jensen & Newburg, 1995; Miraliakbari & Shahidi, 2008). As expected, bovine and almond milk lipids presented a different fatty acid composition (Table 1). More than 400 different fatty acids have been detected in bovine milk (Jensen & Newburg, 1995). Most fatty acids in bovine milk triglycerides were saturated (Table 1). Bovine phospholipids, only 1% of the total lipids, contain mainly long-chain fatty acids with a higher level of unsaturation (Gallier, Gragson, Cabral et al., 2010). Almond lipids contained more unsaturated long-chain fatty acids than bovine lipids and very few short-chain and saturated fatty acids (Table 1). Maguire et al. (2004) reported a ratio unsaturated/saturated of 10.8 for almond fatty acids. By gas chromatography (Table 1), a similar ratio of 10.5 was found. The major bovine fatty acids were myristic, palmitic, stearic and oleic acids, whereas main almond fatty acids were palmitic, oleic and linoleic acids (Table 1). The fatty acid profiles of almond milk and bovine milk (Table 1) were similar to those reported in the literature (Jensen & Newburg, 1995; Miraliakbari & Shahidi, 2008). The values for almond fatty acids reported by previous researchers (Table 1) were determined using gas chromatography by extracting lipids from the seeds without processing into an aqueous suspension. The values for bovine fatty acids from previous researchers (Table 1) were obtained with gas chromatography with capillary columns, however the origin of the milk was not specified. Variations in fatty acid composition of bovine milk lipids arise from different techniques of analysis and extraction, and environmental and animal factors such as feeding, breed and season. 3.1.2. Cholesterol and phytosterols Almond milk and bovine milk contained a similar ratio of total sterols/total lipids (Table 2). Cholesterol accounts for 90% of the total sterols of bovine milk, the rest being in the ester forms (Jensen & Newburg, 1995). Bovine whole milk contains 14 mg cholesterol/ 100 g (Jensen & Newburg, 1995), which is close to the value reported in Table 2. Maguire et al. (2004) and Miraliakbari and Shahidi (2008) reported low amounts of campesterol (0.05 mg/g lipid and 0.09 mg/g lipid respectively) and stigmasterol (0.055 mg/g lipid and 0.19 mg/g lipid respectively) in almonds and a higher amount of b-sitosterol (about 2 mg/g lipid and 2.3 mg/g lipid respectively). Almond milk also contained more b-sitosterol than campesterol and stigmasterol (Table 2). Differences in almond fatty acid composition and phytochemical content from the literature may arise from the use of different cultivars of almonds between studies (Maguire et al., 2004; Miraliakbari & Shahidi, 2008), different techniques of analysis, and different extraction methods, as most of the compositional analyses are carried out on almond oil extracted from the nut (Miraliakbari & Shahidi, 2008).The presence of very low amount of cholesterol in three cultivars of almond has been reported in a recent review (Cherif et al., 2009). 3.1.3. Phospholipid composition The phospholipid composition of bovine milk and almond milk is presented in Table 3. The main almond milk phospholipids were phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylethanolamine (PE), and the main bovine milk phospholipids were PC, PE and sphingomyelin (SM) in similar quantities, and PI and phosphatidylserine (PS) (Table 3). Similar values of bovine milk phospholipids were reported by others (Christie, Noble, & Davies, 1987; Gallier, Gragson, Cabral et al., 2010); however the variations are due to the use of different methods of extraction and analysis. No lysophospholipids or phosphatidylglycerol (PG) were detected using 31P NMR in bovine milk, whereas low amounts of PG, cardiolipin and N-acylphosphatidylethanolamine were detected in almond milk (Table 3).

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S. Gallier et al. / Food Chemistry 132 (2012) 1996–2006 Table 1 Fatty acid composition of almond and bovine milk samples. Fatty acid

Almond milk % FA

Bovine milk % FA

Almond nut g/100 g lipida

Bovine milk wt.%b

C4:0 Butyric C6:0 Caproic C8:0 Caprylic C10:0 Capric C11:0 Undecanoic C12:0 Lauric C13:0 Tridecanoic C14:0 Myristic C14:1n5-cis-9-Myristoleic C15:1n5-cis-10-Pentadecenoic C16:0 Palmitic C16:1n7-cis-9-Palmitoleic C17:0 Margaric C17:1n7-cis-10-Heptadecenoic C18:0 Stearic C18:1n9t Elaidic C18:1n7t Vaccenic C18:1n9c Oleic C18 :1n7c Vaccenic C18:2n6t Linolelaidic C18:2n6c Linoleic C20:0 Arachidic C18:3n6-cis-6,9,12-Gamma linolenic C20:1n9-cis-11-Eicosenoic C18:3n3-cis-9,12,15-Alpha linolenic C21:0 Heneicosanoic C20:2n6-cis-11,14-Eicosadienoic C22:0 Behenic C20:3n6-cis-8,11,14-Eicosatrienoic C22:1n9-cis-13-Erucic C20:3n3-cis-11,14,17-Eicosatrienoic C23:0 Tricosanoic C20:4n6-cis-5,8,11,14-Arachidonic C22:2n6-cis-13,16-Docosadienoic C24:0 Lignoceric C20:5n3-cis-5,8,11,14,17-Epa C24:1n9-cis-15- Nervonic C22:5n3-cis-7,10,13,16,19-DPA C22:6n3-cis-4,7,10,13,16,19-DHA SCFA (C4–C14) MCFA (C15–C17) LCFA (C18–C22) SFA MUFA PUFA

0.83 ND ND ND ND ND ND ND ND ND 5.83 ± 0.03 0.42 ± 0.01 0.08 ± 0.01 ND 1.62 ± 0.05 ND ND 66.58 ± 0.06 1.21 ± 0.01 ND 24.34 ± 0.06 0.07 ND 0.07 ± 0.01 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.83 6.91 92.25 8.66 68.22 23.11

6.06 ± 0.06 5.71 ± 0.25 2.29 ± 0.00 3.85 ± 0.02 0.42 ± 0.42 3.59 ± 0.00 0.10 ± 0.01 11.41 ± 0.01 1.03 ± 0.02 ND 32.79 ± 0.25 1.51 ± 0.03 0.93 ± 0.01 ND 9.52 ± 0.04 0.19 ± 0.01 1.87 ± 0.01 15.99 ± 0.08 0.30 ± 0.02 ND 0.78 ± 0.02 0.19 ± 0.01 ND ND 0.71 ± 0.01 0.64 ± 0.00 ND ND ND ND ND ND ND ND ND ND ND 0.12 ± 0.00 ND 34.44 ± 0.39 35.24 ± 0.29 30.31 ± 0.20 77.49 ± 0.68 21.52 ± 0.17 1.61 ± 0.03

–

4.5 2.3 1.3 2.7 0.3 3.0 0.2 10.7 0.9 0.3 28.2 1.8 1.3 0.4 12.6 – 1.7 21.4 – 0.4 2.9 0.2 2.9 0.6 0.3 – 0.03 – – – – – 0.2 – – – – – – – – – – – –

0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.00 0.00 ± 0.00 – 7.36 ± 0.02 0.66 ± 0.00 0.05 ± 0.00 – 1.56 ± 0.01 – – 60.93 ± 0.03 – – 29.21 ± 0.00 0.06 ± 0.00 0.10 ± 0.00 0.00 ± 0.00 – 0.00 ± 0.00 – 0.00 ± 0.00 – 0.00 ± 0.00 – – – – 0.00 ± 0.00 – – – – – – – 9.09 ± 0.02 61.60 ± 0.02 29.31 ± 0.00

ND: none detected, FA: fatty acid, SCFA: short-chain fatty acid, MCFA: medium-chain fatty acid, LCFA: long-chain fatty acid, SFA: saturated fatty acid, MUFA: monounsaturated fatty acid, PUFA: poly-unsaturated fatty acid. a Venkatachalam and Sathe (2006). b Jensen and Newburg (1995).

Table 3 Phospholipid profiles of almond milk and bovine milk determined by

Table 2 Sterol profile of almond milk and bovine milk. Sterols

Brassicasterol b-Sitosterol Stigmasterol Ergosterol Campesterol Cholesterol Total sterols

Almond milk

Bovine milk

mg/100 g milk

mg/g lipid

mg/100 g milk

mg/g lipid

ND 15.5 ± 1.5 3.0 ± 0.0 ND 1.0 ± 0.0 ND 19.5 ± 1.5

ND 1.52 ± 0.180 0.294 ± 0.006 ND 0.098 ± 0.002 ND 1.91 ± 0.188

ND ND ND ND ND 15.5 ± 2.5 15.5 ± 2.5

ND ND ND ND ND 2.82 ± 0.515 2.82 ± 0.515

31

P NMR.

Phospholipid

Almond milk (mol%)

Bovine milk (mol%)

Phosphatidylcholine Phosphatidylethanolamine N-Acylphosphatidylethanolamine Sphingomyelina Phosphatidylinositol Phosphatidylserine Phosphatidylglycerol Cardiolipin Phosphatidic acid Others

31.9 ± 2.34 6.63 ± 0.63 1.26 ± 0.13 ND 15.8 ± 2.41 ND 1.56 ± 0.02 1.32 ± 0.23 39.3 ± 0.09 2.18 ± 0.55

25.8 ± 0.02 27.4 ± 0.50 ND 27.3 ± 0.75 7.11 ± 0.00 10.2 ± 0.98 ND ND ND 2.24 ± 2.24

ND: non-detected. The detection limit of the technique is 10 mg/100 g, therefore given values below 10 mg/100 g are approximate and minor phytosterols in almond milk were in too small quantity to be detected.

ND: not detected. a Including dihydrosphingomyelin.

Almond oil contains about 0.25% PS, 0.15% PI, 0.2–0.5% PC, and 0.55% sphingolipids (Miraliakbari & Shahidi, 2008). Boukhchina, Sebai, Cherif, Kallel, and Mayer (2004) found a similar amount of

PE and PC in almond lipids whereas Beisson et al. (2001) found different proportions of phospholipids in almond OB with 60% being PC and 12% PE. The variations with the results in Table 3

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arise mostly from the studied material, as the values reported in the literature were obtained after solvent extraction of the almond lipids from the nut (Boukhchina et al., 2004; Miraliakbari & Shahidi, 2008). Almond milk contained a high amount of PA but a low amount of PE (Table 3); this is probably due to the activation of phospholipase D during the soaking of the sliced almonds overnight, as there was no microbial contamination in the almond milk preparation (results not shown). Indeed, soaking of the seeds, which is a common step in soy milk processing for example, induces a phospholipase D-catalysed transphosphatidylation of PC and PE, leading to the production of PA (Boatright & Snyder, 1993; Munnik & Testerink, 2009; Roughan, Slack, & Holland, 1978). Almond milk did not contain any SM whereas SM represented almost a third of bovine milk phospholipids (Table 3); the presence of SM in membranes is a determinant for the occurrence of lipid phase separation at the surface of the membrane and therefore the fluidity of the membrane (Gallier, Gragson, Jimenez-Flores et al., 2010). 3.1.4. Protein composition Almond and bovine milk proteins were determined by tricineSDS–PAGE (Fig. 1). The gel patterns of both milk proteins (Fig. 1) show the proteins present in the serum phase and the proteins adsorbed onto the interface of either the oil bodies or the milk fat globules. Most of the almond proteins belong to the oleosin family (Beisson et al., 2001). Almond oleosins are low-molecular-weight proteins with poor water solubility, due to a long highly hydrophobic domain of about 70 amino acid residues (Beisson et al., 2001). These oleosins are specific to oil bodies and can be solubilised in the presence of SDS. By SDS–PAGE in the presence of b-mercaptoethanol, a reducing agent, up to 25 polypeptides, from 12 to 166 kDa, were detected (Fig. 1; Sathe et al., 2002). More recently,

using two-dimensional electrophoresis, 188 proteins were detected in the seed of almonds (Li & He, 2004); the almond major protein, also called amandin, represents up to 70% of the total soluble proteins (Sathe et al., 2002). Amandin is an allergen and is therefore highly thermostable and resistant to proteolysis (Sathe et al., 2002). Amandin is anoligomeric protein made up of two polypeptides of molecular weight ranging from 20 to 22 kDa (basic b-chain) and 40 to 42 kDa (acidic a-chain) (Fig. 1). Sathe (1993) showed that the polypeptides are linked via disulphide bonds forming an oligomeric protein of 62–66 kDa. Bovine milk contains 36 g/L of proteins, 29.5 g/L being caseins and 6.3 g/L being whey proteins (Swaisgood, 1995). Caseins include as1-, as2-, b-, j- and c-caseins and whey proteins, include a-lactalbumin, b-lactoglobulin (Fig. 1), serum albumin and immunoglobulin. Bovine milk fat globule membrane (MFGM) contains more than 120 proteins (Reinhardt & Lippolis, 2006), the main ones being xanthine oxidase, butyrophilin, adipophilin, PAS 6/7, mucin 1, PAS III and CD36 (Singh, 2006). Bovine MFGM contains more than 25 enzymes (Singh, 2006). Mucins and CD36 stains poorly with Coomassie blue (Mather, 2000) and therefore could not be detected in this study (Fig. 1). 3.2. f-Potential measurements and particle size distribution The surface structure and composition of a particle strongly influence its f-potential. The f-potential of bovine MFG was 36.33 ± 2.18 mV, which agrees with previous work (Ye, Cui, & Singh, 2011), and the f-potential of almond OB was 29.9 ± 1.99 mV. Bonsegna et al. (2011) reported a f-potential of 30 mV for reconstituted almond OB at pH 7. The net negative charges of the oleosins at the surface of the almond OB contribute to their negative f-potential and prevent the coalescence of oil bodies by

Fig. 1. Tricine SDS–PAGE of bovine and almond proteins in milk in the presence of b-mercaptoethanol. (1) Almond milk, (2) bovine milk, (3) high molecular weight standards, (4) low molecular weight standards. PP: polypeptide.

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OB (3.47 ± 0.13 lm and 2.62 ± 0.12 lm respectively), and therefore the specific surface area of almond milk OB (2.28 ± 0.10 m2/g) was greater than the area of bovine MFG (1.67 ± 0.03 m2/g).

electrical repulsion (Huang, 1994). The outer layer of the bovine MFGM is dominated by the protruding glycocalyx and peripheral proteins (Singh, 2006). Therefore the proteins at the surface of the OB and MFG determined their negative charge at neutral pH. Fig. 2 shows the particle size distribution of almond milk OB and bovine MFG. The size distributions of almond milk OB and bovine MFG were monomodal, ranging from 0.5 to 13 lm and bimodal, ranging from 0.5 to 15 lm, respectively (Fig. 2). Bovine MFG are larger in size than almond milk OB (Fig. 2). The span of the size distribution was lower for bovine MFG (1.20 ± 0.05) than for almond OB (1.44 ± 0.02). The volume-weighted average diameter, d43, is sensitive to the presence of large particles, whereas the surface-weighted average diameter, d32, is more sensitive to the presence of small particles (Couvreur & Hurtaud, 2007). The parameters of size distribution of bovine MFG and almond OB were consistent with results from Couvreur and Hurtaud (2007) and Beisson et al. (2001) respectively. Bovine MFG presented a larger d43 (4.62 ± 0.07 lm) and d32 (3.57 ± 0.06 lm) than almond milk

3.3. Structure of the almond milk OB and bovine MFG using different fluorescent probes and CLSM Almond and bovine milk samples were stained with a lipophilic dye and a dye that attached to proteins (Fig. 3). Nile Red exhibits an intense fluorescence in a lipid environment, due to its solubility in neutral lipids (Greenspan, Mayer, & Fowler, 1985). Nile Red stained similarly the lipid core of the almond milk OB and bovine MFG (Fig. 3). Fast green FCF is a dye electrostatically attracted to charged groups on proteins (Merril & Washart, 1998). Fast Green FCF also stained similarly the proteins at the surface of the OB and MFG (Fig. 3). The surface of the almond milk OB was entirely covered by proteins (Fig. 3 zoom), some of them being oleosins as described by Huang (1994). In the almond milk micrograph (Fig. 3), no protein

14 12

Volume (%)

10 8 6 4 2 0 0

2

4

6

8

10

12

14

16

18

Lipid droplet diameter (µm) Fig. 2. Droplet size distribution of almond milk oil bodies ( ) and bovine milk fat globules ().

Fig. 3. CLSM two-dimensional (2D) images of almond milk oil bodies (left) and bovine milk fat globules (right) stained with Nile Red and Fast Green FCF. The left image includes a zoom (top left corner) on one almond oil body showing the protein layer covering the entire surface of the lipid core. Scale bars = 25 lm (large image) and 5 lm (zoom). (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. CLSM 2D images of almond milk oil bodies (left) and bovine milk fat globules (right) stained with Rd-DHPE (A and B), with Rd-DHPE and Con A (C and D), and with RdDHPE and WGA (E). No emission from WGA was observed after staining of almond milk oil bodies with WGA and Rd-DHPE. The white arrows are pointing at liquiddisordered domains (dark area by contrast with the red area, the liquid-disordered domains) on the surface of the milk fat globule membrane. Scale bars = 10 lm (A, B, D and E) and 25 lm (C). (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

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bodies were observed, in contrast to a study by others (Young, Schadel, Pattee, & Sanders, 2004) using transmission electron microscopy to study the structure of the almond cotyledon. The membranes of almond milk OB and bovine MFG were stained with a fluorescent phospholipid analogue to observe the lateral phospholipid distribution (Fig. 4A and B). Phospholipids are the backbone of biological membrane structure. The coexistence of a liquid-ordered phase (arrows in Fig. 4B), rich in sphingomyelin and cholesterol, with a liquid-disordered phase, rich in unsaturated phospholipids, within the bovine MFGM was observed by Gallier, Gragson, Jimenez-Flores et al. (2010). It is not surprising to observe the absence of a liquid-ordered phase in the almond OB membrane (Fig. 4A), as cholesterol (Quinn & Wolf, 2009) and most importantly sphingomyelin (Brown & London, 1998), which are necessary for phase separation, were largely absent or at very low concentrations in almond lipids (Tables 2 and 3; Cherif et al., 2009). Almond and bovine milk samples were stained with the lectins Con A (Fig. 4C and D) and WGA (Fig. 4E), to observe the distribution of glycosylated molecules at the surface of the lipid droplets. Con A allows the location of glycoproteins and glycolipids containing a-mannopyranosyl and a-glycopyranosyl residues, whereas WGA allows the location of glycoproteins and glycolipids with N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues (Molecular Probes Handbook; www.probes.invitrogen.com). Therefore they bind to bovine MFGM glycoproteins, such as butyrophilin, CD36, the mucins MUC1 and MUC15 and PAS6/7, and gangliosides (Singh, 2006). Bovine MFGM glycoproteins and glycolipids were heterogeneously distributed at the surface of the MFG and absent from the liquid-ordered phase of the membrane (Fig. 4D and E). A complete review on the localisation of the MFGM proteins was recently published (Vanderghem et al., 2011). The almond OB membrane seemed to contain very low levels of glycoproteins and glycolipids, as little (Fig. 4C) to no (results not shown) staining was observed with the lectins ConA and WGA, respectively. As reported by Sathe et al. (2002), amandin, the major almond protein, is not a glycoprotein, however some of the minor almond proteins are glycoproteins (Taga, Waheed, & Vanetten, 1984). An attempt was made to stain simultaneously almond milk OB with Rd-DHPE and Fast green FCF. Unfortunately, as the almond OB membrane is very thin and under the microscope resolution limit, it was not possible to differentiate the phospholipid layer from the protein layer (results not shown). 3.4. Chemical fingerprint of almond milk OB and bovine MFG by confocal Raman microscopy Both almond milk OB and bovine MFG presented variations in lipid composition according to their size (Figs. 5 and 6). More information was available from large lipid droplets, due to higher concentration of all components and the 1-lm resolution limit of the microscope. Using a model (Forrest, 1978) to determine the percentage of unsaturation in lipids and an averaged ratio I1654/I1742 (Fig. 6), almond milk lipids contained 87.8% unsaturated fatty acids, which is close to the values reported in Table 1, and bovine milk lipids contained 42.3% unsaturated lipids, which is close to the value of 39 ± 5% reported by Forrest (1978) but twice as much as the value in Table 1, probably due to the undetectable level of triglycerides in the globules smaller than 1 lm in size with confocal Raman microscopy. The band at 1742 cm 1 is related to the C@O stretching of the ester groups of triglycerides (Krafft, Neudert, Simat, & Salzer, 2005). The CH2 deformation vibrations at 1443 cm 1 are specific to saturated fatty acids, whereas the C@C stretching vibrations at 1654 cm 1 are specific to unsaturated fatty acids in the cis configuration. The two ratios I1654/I1443 and I1654/I1742 are indicative of

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the degree of unsaturation of the samples (Forrest, 1978; Ozaki, Cho, Ikegaya, Muraishi, & Kawauchi, 1992). The results in Fig. 6 were in agreement with our previous work (Gallier et al., 2011), though the ratio I1654/I1744 in Fig. 6 was slightly higher than the same ratio for MFG (Gallier et al., 2011). The ratios I1654/I1443 and I1654/I1742 revealed that bovine milk fat globules had a low degree of unsaturation, whereas almond milk OB had a high degree of unsaturation. In addition, this indicated that the former has a low iodine value and the latter a high iodine value, as the ratio I1654/I1443 and the iodine value are linearly correlated (Ozaki et al., 1992). Indeed, Moayedi et al. (2011) reported an iodine value of 90–96 g/g of almond oil, whereas butter has an iodine value of 31 g/g of fat (Ozaki et al., 1992). The CH in-plane bending modes of ethylene groups give a band at 1262 cm 1. The Raman spectra of almond OB showed a stronger peak at 1262 cm 1 than bovine MFG (Fig. 5A and B), this again is related to the higher level of unsaturation of almond lipids (Ozaki et al., 1992). Cis and trans C@C bonds in unsaturated fatty acids give stretching bands around 1650–1660 and 1670–1680 cm 1, respectively. Only the band at 1654 cm 1 from cis unsaturated fatty acids was observed in Fig. 5A and B for almond and bovine lipids. The Raman spectra of almond lipids presented a stronger peak at 3007 cm 1 due to cis unsaturated @CH stretching modes (Fig. 5C and D). C–C mobility of the hydrocarbon chains is determined by the intensities of the bands at 1065, 1080 and 1130 cm 1, and the C– H mobility by the ratio of the peaks at 2850 and 2885 cm 1 (Forrest, 1978). Both almond and bovine lipids were mostly in a fluid state at the experimental temperature (22 °C) (Figs. 5 and 6). However, almond lipids were more mobile than bovine lipids, as indicated by the greater intensity of the peak at 1080 cm 1 relative to the peaks at 1065 and 1130 cm 1 (Fig. 5A and B), which is associated with the increase in gauche rotamers (Ozaki et al., 1992). The band at 1130 cm 1 (out-of-phase C–C solid fat) was not detected in the Raman spectra of almond lipids but was observed in the Raman spectra of bovine lipids (Fig. 5A and B), denoting that some of the bovine lipids are in a crystalline state at the experimental temperature. Bovine milk lipids contained a larger diversity of fatty acids and a higher ratio of saturated and short-chain fatty acids (Table 1), therefore presented a higher melting point than almond lipids, and each fatty acid is, at a given temperature, in a different state, due to variations in chain length and level of unsaturation. The region from 2600 to 3200 cm 1 contains information about the mobility and structure of the hydrocarbon chains of lipids (Forrest, 1978). The stronger intensity of the peak at 2850 cm 1 relative to the peak at 2885 cm 1 indicates higher mobility of the acyl chains (Forrest, 1978). The Raman spectra of almond lipids presented an extra band at 2895 cm 1 (Fig. 5C), related to the anti-symmetric CH stretches of the methylene groups (Snyder, Strauss, & Elliger, 1982), which made the evaluation of the peak intensity of the band at 2885 cm 1 more difficult. The ratio I2935/ I2885 gives information about inter-chain order and gauche conformation along the hydrocarbon chains; however it is difficult to measure the intensity of the band at 2935 cm 1, related to CH3 symmetric stretching vibrations, as this band is a combination of the Fermi resonance components at 2922 cm 1 (polymethylene chain) and at 2938 cm 1 (methyl group) (Snyder et al., 1982). Even at low concentration, carotenoids can be detected with confocal Raman microscopy by resonance enhancement of the carotenoid Raman bands (Gallier et al., 2011). Vitamin A and b-carotene represent only 0.1% of bovine lipids (Forrest, 1978). Almonds contain 5 IU/100 g vitamin A and 3 lg/100 g b-carotene (Chen et al., 2006). Less carotenoid signal (bands at 1006, 1158 and 1526 cm 1) was observed from the bovine MFG (Fig. 5B) than observed in our previous work (Gallier et al., 2011). This is mainly because the bovine milk in the present study originated from Friesian cows, whose milk contains a lower amount of carotenoids

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Fig. 5. Raman spectra of almond milk oil bodies (left) and bovine milk fat globules (right) of size 2 lm (a), 4 lm (b), 8 lm (c), 10 lm (d), 14 lm (e) (left figures), 13 lm (e) (right figures) in the region 600–1800 cm 1 (A and B) and in the CH region 2600–3200 cm 1 (C and D). (a.u.: arbitrary units).

(Newstead, 1976) than Jersey and crossbred cows’ milk used in our previous work. However weak C@C and C–C stretching bands at 1526 and 1158 cm 1, respectively, were observed in the bovine MFG larger than 10 lm in size, with increasing intensity with increasing size (Fig. 5B). This denoted an increase in carotenoid concentration with increasing size of the globules. The carotenoid bands were not detected in the spectra of almond lipids (Fig. 5A). The Raman spectra of almond lipids presented a broader and stronger band around 803 cm 1 and a stronger one at 598 cm 1 than bovine lipids (Fig. 5A and B). Vitamin E contains a chromanol

ring, and cholesterol and phytosterols a multiple-ring structure (Cherif et al., 2009; Walstra et al., 2006). Almond lipids contained more sterols (Table 2) and more vitamin E, about 0.45 mg/g lipid (Maguire et al., 2004), than bovine lipids, about 0.1 mg vitamin E/ 100 g of milk (Walstra et al., 2006). Therefore the band at 803 cm 1 could be due to the breathing mode of the aromatic rings of tocopherol and sterols (Parker, 1983). Another hypothesis is that this band could be due to the Fermi resonance of the phenolic group of tyrosine (Spiro & Gaber, 1977) from the oleosin domain anchored in the triacylglycerol as described by Huang (1994). The band at

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Relative intensity (a.u.)

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Lipid droplet diameter (µm) Fig. 6. Qualitative evaluation of the liquid/crystal fat ratio and unsaturation level of almond milk oil bodies and bovine milk fat globules with varying size. The columns indicate the CH2 anti-symmetric stretching band (2885 cm 1, acyl chains in crystalline state) intensities (I) relative to the CH2 symmetric stretching band (j bovine, almond, 2850 cm 1, acyl chain in liquid state) intensities, or the double stretching band (1654 cm 1) intensities relative to either the CH2 scissoring band ( bovine, almond, 1443 cm 1) intensities or the triglyceride band ( bovine, almond, 1742 cm 1) intensities. There was no detectable level of triglycerides in bovine milk fat globules of 1 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

598 cm 1corresponds to the vibration of the glycerol backbone of sterol and triglyceride molecules (Krafft et al., 2005). With all the differences stated above, confocal Raman microscopy could be used to differentiate between several types of oils and fats. 4. Conclusions This study revealed and confirmed the differences in chemical, physical and structural properties between a plant-derived source of lipid, the almond oil body, and an animal source of lipid, the bovine milk fat globule. Almond OB contained mostly long-chain unsaturated fatty acids, phytosterols, nosphingomyelin, and a major almond protein. Bovine MFG were rich in saturated fatty acids and cholesterol, larger in size, and richer in sphingomyelin and phosphatidylethanolamine. Their membranes differed in composition and structure, and only the bovine MFGM showed liquid-ordered domains at its surface. Variations in composition and structure will most likely lead to different ways of digestion and absorption upon milk or nut consumption. Ongoing study of the digestion of these two types of lipids is being carried out in our laboratory. Acknowledgements The authors would like to thank Dr. Anwesha Sarkar (Nestlé Research Center, Lausanne, Switzerland) for helpful preliminary discussions, and Jacqui Kao (University of Otago, Dunedin, New Zealand) for her technical assistance with the confocal Raman microscope. The work was supported by a Centre of Research Excellence fund from the Tertiary Education Commission and the Ministry of Education, New Zealand. References Beisson, F., Ferte, N., Bruley, S., Voultoury, R., Verger, R., & Arondel, V. (2001). Oilbodies as substrates for lipolytic enzymes. Biochimica Et Biophysica ActaMolecular and Cell Biology of Lipids, 1531(1–2), 47–58. Boatright, W. L., & Snyder, H. E. (1993). Soybean protein bodies: phospholipids and phospholipase D activity. Journal of the American Oil Chemists Society, 70(6), 623–628.

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