Elsevier

International Dairy Journal

Volume 18, Issue 12, December 2008, Pages 1081-1089
International Dairy Journal

Heterogeneity of milk fat globule membrane structure and composition as observed using fluorescence microscopy techniques

https://doi.org/10.1016/j.idairyj.2008.06.001Get rights and content

Abstract

Fluorescence microscopy studies of fat globules in milk and milk products using fat-soluble stains (Nile Blue or Nile Red) allow visualisation of the neutral fat (core) of the globules, but provide no information about the milk fat globule membrane (MFGM). We applied the lipophilic probes 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid (DiIC18(3)-DS) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), as well as fluorescent conjugates of the lectin wheat germ agglutinin (WGA488, WGA594 and WGA647), to milk to stain specifically the MFGM in its native environment. Using fluorescence microscopy, we observed various distribution patterns of the probes. This demonstrates that the MFGM of fat globules in harvested milk is structurally and chemically heterogeneous both within and among globules from the same species and probably between species. We propose that the use of membrane-specific fluorescent probes has significant potential for providing real time structural and chemical information about the MFGM in matrices such as mammary gland tissue, harvested milk, and milk products.

Introduction

Fat globules in milk are surrounded by a thin layer of surface-active material, which is called the milk fat globule membrane (MFGM). Upon secretion of the fat droplet from the mammary secretory cell, the structure of the MFGM is trilaminar, consisting of a surface-active inner layer that surrounds the intracellular fat droplet, then a dense proteinaceous coat and finally a true bilayer membrane originating from the secretory cell apical plasma membrane (Keenan & Mather, 2002). Following secretion of the fat globule by the secretory cell, the MFGM is believed to change to some degree, e.g., by partial loss of the bilayer membrane, although the extent of the loss is unclear (Evers, 2004a).

Much of the current knowledge of the structure and molecular organisation of the MFGM has been obtained using morphological techniques such as (immuno)electron microscopy applied to fat globules that had been isolated from the original milk sample (Buchheim, 1982, Franke et al., 1981, Henson et al., 1971, Horisberger et al., 1977, Mather et al., 2001, Monis et al., 1975, Pinto da Silva et al., 1980, Robenek et al., 2006, Sasaki and Keenan, 1979, Schmidt and Buchheim, 1992, Welsch et al., 1988, Wooding, 1971). We were particularly intrigued by recently published freeze-fracture electron microscopy results (Robenek et al., 2006), which revealed variability in the physical structure of the surface of the MFGM of individual globules. However, electron microscopy results apply to fat globules that may, or may not, have undergone substantial physical and/or chemical modification, and the environment of which has been drastically changed. Hence, the degree to which the results represent the structure of the membranes of fat globules in the original sample remains uncertain.

Fluorescence microscopy can be used to study fat globules without isolating them from the milk sample matrix. The commercially available fluorescent dyes Nile Blue and Nile Red have been used to study fat globules in milk and milk products, using either conventional fluorescence microscopy or confocal laser scanning microscopy (CLSM; note that this abbreviation is also used for the confocal laser scanning microscope, as defined by the context) (Everett and Olson, 2003, Evers, 2004b, Herbert et al., 1999, Lopez and Briard-Bion, 2007, Lopez et al., 2008, Lopez et al., 2007, Michalski et al., 2004, Sutheerawattananonda et al., 1997, Trachoo, 2003, Ye et al., 2005). However, these neutral fat dyes stain the fat core of the globules and, therefore, provide no information about the MFGM. The fluorescent dye Acridine Orange has been used to visualise fat globules (King, 1958, Scolozzi et al., 2003) or cytoplasmic crescents associated with fat globules (Janssen and Walstra, 1982, Patton and Huston, 1988), but this dye is presumed to stain cytoplasmic matter rather than the MFGM itself (Scolozzi et al., 2003). Immunofluorescence techniques have revealed generally patchy distributions of butyrophilin and other membrane proteins (McManaman, Palmer, Wright, & Neville, 2002), but these techniques are known to introduce artefacts (Robenek et al., 2006).

It is in this context that we sought to develop new techniques that would specifically probe, in real time, the structural and compositional details of the MFGM in its native environment (i.e., milk) in a non-destructive manner. Thus, a range of commercially available fluorescent probes, individually and in combination, were screened by means of conventional fluorescence microscopy and CLSM. In this paper, we present staining patterns of the MFGM by two lipophilic probes, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid (DiIC18(3)-DS) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64), and by the lectin wheat germ agglutinin (WGA).

Section snippets

Chemicals

Reverse osmosis water was purified using a Milli-Q system (Millipore Corporation, Bedford, MA, USA). Fluorescent probes (solids for reconstitution; molecular structures given in Fig. 1) were obtained from Molecular Probes (Eugene, OR, USA): DiIC18(3)-DS, FM4-64 and wheat germ agglutinin (WGAxyz, where xyz indicates the particular Alexa Fluor® conjugate, e.g., WGA488). Single-probe stock concentrations were 1 mg mL−1 Milli-Q water, unless indicated otherwise. A dual-probe stock solution contained

Lipophilic probes

An ethanolic DiIC18(3)-DS stock solution immediately resulted in a small proportion (estimated <5%) of the fat globules being brightly stained. Subsequent incubation at room temperature for several hours resulted in the staining of further fat globules, but not as brightly as those stained initially. DiIC18(3)-DS was located exclusively, but often heterogeneously, in the MFGM (Fig. 2). Because of the risk of ethanol perturbing the membrane structure by denaturing membrane proteins, aqueous

WGA controls

Conflicting results for WGA control studies using GlcNAc have been reported in the literature. In washed bovine milk fat globules and human milk fat globules, GlcNAc appeared to completely inhibit WGA binding (Farrar, Harrison, & Mohanna, 1980). Similarly, non-specific binding of WGA in the presence of N-acetylchitopentaose was reported to be very low (Horisberger et al., 1977). However, in equine milk, GlcNAc had no inhibiting effect (Welsch et al., 1988).

To our knowledge, no studies of WGA

Conclusions

Fluorescence microscopy using MFGM-specific probes has revealed chemical and structural heterogeneity at the micron and submicron scale in the MFGM, not only of individual globules but also among globules. In providing chemical as well as physical information on MFGMs in their native environment, our results confirm, complement and substantially extend previously reported morphological results for the MFGM of fat globules isolated from milk. The techniques developed show significant potential

Acknowledgements

We thank Elizabeth Nickless for support with CLSM. We thank Massey University No. 1 Dairy Farm staff, Kingsmeade sheep farm staff and the human volunteer for providing milk samples. This research was funded in part by the New Zealand Tertiary Education Commission through a Bright Future scholarship (J. M. Evers). Dr T. Coolbear is acknowledged for critical evaluation of this manuscript.

References (49)

  • C. Lopez et al.

    Development of the milk fat microstructure during the manufacture and ripening of Emmental cheese observed by confocal laser scanning microscopy

    International Dairy Journal

    (2007)
  • S. Patton et al.

    Incidence and characteristics of cell pieces on human milk fat globules

    Biochimica et Biophysica Acta

    (1988)
  • M. Sasaki et al.

    Ultrastructural characterization of carbohydrate distribution on milk lipid globule membrane

    Cell Biology International Reports

    (1979)
  • M. Sutheerawattananonda et al.

    Fluorescence image analysis of process cheese manufactured with trisodium citrate and sodium chloride

    Journal of Dairy Science

    (1997)
  • F.B.P. Wooding

    The structure of the milk fat globule membrane

    Journal of Ultrastructure Research

    (1971)
  • W. Buchheim

    Paracrystalline arrays of milk fat globule membrane-associated proteins as revealed by freeze-fracture

    Naturwissenschaften

    (1982)
  • W. Buchheim

    Membranes of milk fat globules – ultrastructural, biochemical and technological aspects

    Kieler Milchwirtschaftliche Forschungsberichte

    (1986)
  • W. Buchheim et al.

    Glycoprotein filament removal from human milk fat globules by heat treatment

    Pediatrics

    (1988)
  • W. Buchheim et al.

    Electron microscopy and carbohydrate histochemistry of the human milk fat globule membrane

  • S. Danthine et al.

    Évolution des connaissances sur la membrane du globule gras du lait: synthèse bibliographique

    Lait

    (2000)
  • Evers, J. M. (2008). Novel analytical techniques for studying the milk fat globule membrane. Ph.D Thesis, Massey...
  • W.W. Franke et al.

    Antibodies to the major insoluble milk fat globule membrane-associated protein: specific location in apical regions of lactating epithelial cells

    Journal of Cell Biology

    (1981)
  • S. Herbert et al.

    Multiple fluorescence labelling of proteins, lipids and whey in dairy products using confocal microscopy

    Lait

    (1999)
  • M.M.T. Janssen et al.

    Cytoplasmic remnants in milk of certain species

    Netherlands Milk and Dairy Journal

    (1982)
  • Cited by (0)

    View full text