Microstructural Analysis

2.4.2.1 Microscopy

Microscopes are used as a visual technique to analyse the microstructure of food products (Auty, 2002). They provide a powerful tool in assisting in the understanding of the relationship between physico-chemical changes in a system and the texture of the product (Everett & Auty, 2008). The main microscope techniques applied to the study of dairy products are compound light, stereo, confocal, scanning electron and transmission electron microscopes (Auty, 2002).

2.4.2.1.1 Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is a microscopy technique that offers a method of observing a sample without disturbing its internal structure (Everett, Ding, Olson, & Gunasekaran, 1995a; Joshi, Muthukumarappan, et al., 2004a). The confocal microscope was invented in 1955 by Minsky (Minsky, 1988). Confocal refers to the common focal point of the image of the illumination pinhole and the back projection of the detection pin-hole (Everett et al., 1995b). The laser scanning allows the penetration of the surface of a sample and the visualisation of thin optical sections (Joshi, Muthukumarappan, et al., 2004a; Lopez, Camier, & Gassi, 2007). It has the ability to make selective observations of the optical sections without being affected by the image of out- of-focus regions above and below the plane (Everett et al., 1995a). Due to this optical sectioning it allows the accurate assessment of fat globules and the protein network in its 3-dimensional structure (Hassan, Frank, & Corredig, 2002). This is done by building up a number of adjacent planes to reconstruct a 3-D image of the sample (Everett et al., 1995b).

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Other advantages of the CLSM, other than its ability to observe the internal structure without altering it (Joshi, Muthukumarappan, et al., 2004a), include: reduced preparation time due to samples not needing fixation or dehydration; the ability to continuously monitor a sample; and the ability to reconstruct the 3-D microstructure of the sample (Auty, Fenelon, Guinee, Mullins, & Mulvihill, 1999). Another advantage is the ability to fluorescently stain specific components within a sample (Heertje, Vandervlist, Blonk, Hendrickx, & Brakenhoff, 1987).

CLSM has been used extensively to study the microstructure of dairy products including milk protein gelation (Auty et al., 1999; de Kruif et al., 1995), sodium caseinate emulsions (Dickinson, Radford, & Golding, 2003; Ye & Singh, 2001), model cheese systems (Floury et al., 2009; Smith, Carr, Golding, Reid, & Zhang, 2011; Trivedi et al., 2008), feta cheese (Hassan et al., 2002), cream cheese (Fenoul, Denmat, Hamdi, Cuvelier, & Michon, 2008), Emmental cheese (Lopez, Camier, et al., 2007) and many others.

CLSM have been shown to be a useful technique in studying the microstructure of Mozzarella during the processing and ripening as it enables the imaging of the distribution of fat and protein (Auty, Twomey, Guinee, & Mulvihill, 2001; Hassan et al., 2002). It has also been used in numerous studies to assess the microstructure of Mozzarella including: work illustrating the effect of calcium (Guinee, Feeney, Auty, & Fox, 2002; Joshi, Muthukumarappan, et al., 2004a), pH (Guinee et al., 2002), the addition of phospholipase (Lilbæk et al., 2006) and the effect of storage (Auty et al., 2001).

2.4.2.1.2 Scanning Electron Microscopy

Electron microscopes offer a high resolution technique for studying the microstructure of a material (Everett et al., 1995b; Joshi, Muthukumarappan, & Dave, 2003a). Electron microscopy consists primarily of an electron gun encased in a high vacuum (Auty, 2011).

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The electron beam is focused on the sample with electromagnets (Auty, 2002). In scanning electron microscopy (SEM) the image is created by electrons impinging on the surface of the sample and emitting secondary electrons (Auty, 2011). These secondary electrons are collected to form a topographic image of the sample (Aguilera & Bouchon, 2008; Auty, 2002). The first SEM was developed in 1937 by Von Ardenne (1937) following the work by Knoll in 1935.

One of the key disadvantages of electron microscopy is the sample preparation that is involved, including dehydration, which can lead to artefacts (Everett et al., 1995b; Kalab, 1984; Liboff, Goff, Haque, Jordan, & Kinsella, 1988). The dehydration of samples is needed as high vacuums are used to ensure a clear path for electrons (Aguilera & Bouchon, 2008). Another limitation of microscopy methods is the limited field of veiw that can be observed.

There are a number of different types of SEM, most notably conventional high vacuum SEM where a dried sample is examined and cryo-SEM where a sample is examined when frozen to below -80°C (Kaláb, Allan-Wojtas, & Miller, 1995). However, this project will focus on the use of conventional high vacuum SEM due to the access to equipment.

SEM has been used to illustrate the structure of many different dairy products including: casein micelles (Auty, 2011; Horne, 2002), yogurt (Auty, 2011; Lucey, 2007), spray dried milk powder (Auty, 2002), Cheddar cheese (Hall & Creamer, 1972), and Emmental cheese (Lopez, Camier, et al., 2007).

SEM has been used to study the microstructure of Mozzarella cheese during manufacture (Kiely et al., 1992; Oberg, McManus, & McMahon, 1993). It has also been used as a tool to assess changes within the protein structure during maturation (McMahon et al., 1999) and the effect of freezing on the structure of the cheese (Kuo & Gunasekaran, 2003). SEM has also been used to evaluate the effect of a number of processing and compositional levers on the structure of Mozzarella including: the effect of high pressure microfluidization

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(Tunick, Van Hekken, Cooke, Smith, & Malin, 2000), the effect of calcium (Joshi, Muthukumarappan, et al., 2004a), how low fat Mozzarella compares to LMPS (Tunick, Mackey, et al., 1993), and the investigation into the role of salt in the structure of non-fat Mozzarella (Paulson et al., 1998). This is due to its ability to produce a detailed image of the protein and channel structure within the cheese.

Both confocal and scanning electron microscopy allow different detailed examination of the internal structure Mozzarella, indicating that both techniques would be beneficial in this study in conjunction with other tools.

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