Cheese structure and current methods of analysis

Cheese structure and current methods of analysis

Cheese structure and current methods of analysis

1 International Dairy Journal 18(7):759-773 (2008). 1 2 Cheese structure and current methods of analysis 3 4 David W. Everett a,b, *, Mark A.E. Auty b 5 a Department of Food Science, University of Otago, Dunedin 9054 New Zealand 6 b Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland 7 8 * Corresponding author. Tel.: +64-3-479-7545; fax: +64-3-479-7567 9 E-mail address: (D.W. Everett) 10 11 Abstract 12 13 Important milestones in the understanding of cheese structure are highlighted. 14 The development of complex instrumentation, such as transmission, scanning, cry- 15 scanning and environmental electron microscopy, dynamic oscillatory low-strain 16 rheology, confocal laser scanning microscopy, dynamic light scattering, and nuclear 17 magnetic resonance have facilitated the development of structural models that can 18 be used to predict functional properties.

More recent developments in 19 instrumentation have shown how the components interact and change during 20 cheese ripening. The effects of pasta filata processing, high pressurisation of milk, 21 freezing of cheese, and milk homogenisation on cheese structure and functionality 22 are outlined. Future trends in cheese structural research are predicted. 23 24 Keywords: Cheese structure; Microscopy; Pasta filata; Rheology; Fat; Protein 25 26 1. Introduction 27 28 Over thousands of years cheese has developed from a high moisture, low 29 acid, unsalted fresh curd with a very short shelf life to the more stable product of 30 today.

Shelf-life stability has been improved by the addition of well-characterized 31 cultures, purified rennet solutions or pastes, salt, and a more rigorous 32 understanding of the impact of manufacturing and storage conditions on cheese 33 texture and flavour. As a result, overall quality has improved and variability has 34 decreased for large-scale commercial products. Cheese was historically 35 manufactured as a farmhouse product on a small scale; it is now produced on a 36 large scale, either for consumption as a food on its own or as a food ingredient. 37 The functional properties of cheese ingredients are dictated by the structure, 38 therefore knowledge of how cheese structure is produced and develops during 39 ripening is of great importance to the cheese manufacturer.

These functional 40

Cheese structure and current methods of analysis

2 properties are particularly important for pizza-style cheeses, and include stretching, 41 melting, browning, free oil development and expressible moisture, amongst other 42 attributes. The prediction and subsequent control of these properties requires an 43 understanding of where the components of cheese are located in relation to each 44 other, and how they interact and change during ripening. This review will highlight 45 some of the more important historical developments in our understanding of cheese 46 structure, and the resultant impact upon cheese functionality. A more 47 comprehensive review of cheese structure is provided by Everett (2007).

48 49 1.1. Cheese manufacture 50 51 The production of cheese requires the coagulation of milk, in most cases 52 through the action of chymosin on the κ-casein steric stabilizing layer of the casein 53 micelle (see review by Fox in this issue). Immunogold labelling of antibodies to κ- 54 casein coupled with electron microscopy has shown that this protein is 55 predominantly located on the surface of the micelle (Schmidt & Buchheim, 1992). 56 These observations support the development of the hairy casein model (Holt & 57 Horne, 1996), thus providing fundamental knowledge of how chymosin acts upon 58 the micelle in milk to produce cheese curd.

59 Cheese manufacture is essentially the dehydration of milk in combination with 60 other preservative effects such culturing, acidification, salting, packaging, and 61 refrigeration. The rennet-induced milk coagulum is cut and heated to expel moisture 62 in a process termed syneresis. Curds are later drained, salted, and packaged into 63 fresh cheese. The pH continually drops throughout the process to a value between 64 4.6 and 6.0 for most varieties of cheese. An additional step is added during curd 65 draining for Cheddar-like cheese varieties. In this process, called cheddaring, the 66 pH is allowed to drop over a period of 1-2 h with frequent turning and stacking of 67 curd blocks, after which the curd develops a chicken breast texture (Kaláb & 68 Emmons, 1978) that flows under the weight of the curd blocks.

Electron microscopy 69 has shown quite clearly how the various components in cheese, such as protein 70 fibres, fat, water channels, microbial cells and precipitated minerals interact (Kaláb 71 & Harwalkar, 1973; Kaláb, 1979b), although the sample preparation techniques can 72 introduce artefacts, primarily through the removal of water. 73 74 2. Instrumentation 75 76 Texture is a subjective term that describes sensory perception. Rheology is a 77 method of quantifying texture, however there is often a poor relationship between 78 these two parameters. Despite this, rheology has proved useful for indirectly 79 probing the interactions of cheese structural components, and along with other 80 techniques, has allowed the development of mechanisms to explain functional 81 properties.

Both rheological parameters and texture are influenced by factors that 82

3 include casein-casein, casein-water, and casein-fat interactions, the state of water 83 (bulk, or bound to the casein matrix), pH and the state of calcium (ionic or bound to 84 the casein matrix), temperature, sodium chloride content, and extent of proteolysis 85 (see section 4). 86 Instrumentation has developed rapidly since the 1940s to provide greater 87 insight into cheese texture and structure. These include transmission and scanning 88 electron microscopy (TEM and SEM), confocal laser scanning microscopy (CLSM) 89 in the late 1980s, dynamic oscillatory rheometry, and atomic force microscopy, 90 although the latter technique has not largely been applied to cheese structural 91 studies.

Other more indirect methodologies have provided important information 92 about cheese functionality, despite requiring less expensive equipment, such as 93 measurement of free oil formation, the state of water (bulk, or bound to casein), 94 colour determination, and large-scale deformation texture profile analysis. 95 96 2.1. Microscopy 97 98 Microscopy allows direct visualisation of cheese structure. It is employed as a 99 powerful tool for understanding relationships between textural properties and 100 physico-chemical analyses, for example, observing the state of fat (emulsified or 101 free pools of oil) after heating and stretching pasta filata-style cheese.

102 Confocal laser scanning microscopy offers several advantages over 103 conventional optical light microscopy, including improved resolution (~0.2 µm), the 104 ability to optically section down into the sample to avoid sample cutting artefacts at 105 the surface, assembly of two-dimensional micrographs to form a three-dimensional 106 image by computerised image analysis, and fluorescent labelling of different 107 components within the food structure to examine interactions and relative locations 108 within the food product (Heertje et al., 1987; Auty et al., 2001a). Studies using 109 CLSM have examined the fat globule structure in cheese (Muthukumarappan et al., 110 1995; Gunasekaran & Ding, 1999), milk gelation and cheese melting (Auty et al., 111 1999), permeability of rennet casein gels (Zhong et al., 2004), the effect of the pasta 112 filata process on fat globule coalescence in Mozzarella cheese (Rowney et al., 113 2003b) and Cheddar cheese (Everett et al., 1995; Everett & Olson, 2003), 114 localisation of probiotic bacterial cells (Auty et al., 2001b) and starter cells in cheese 115 (Hannon et al., 2006), location of exopolysaccharides in cheese (Hassan et al., 116 2002), and correlation with sensory data of acid milk gels (Pereira et al., 2006).

Non- 117 confocal fluorescence microscopy with dual-labelling of the fat and casein 118 components has shown the importance of casein hydrolysis and fat in ripening 119 Camembert cheese (Yiu, 1985). Two-photon molecular excitation microscopy offers 120 advantages over standard CLSM: photo-bleaching is confined to the focal plane 121 and optical sectioning can be done to greater depths. This method has been used 122 to track calcium diffusion in cells over time (Soeller & Cannell, 1999), therefore it 123 may find efficacy in examining the slow process of calcium solubilisation and 124 release of calcium ions into the bulk water phase during cheese ripening.


4 Scanning and transmission electron microscopy have proved useful in 126 examining the small-scale structural elements in cheese, such as microorganisms 127 and the casein matrix, and in particular, changes that occur in the matrix during the 128 manufacturing and ripening process (Kimber et al., 1974; Kaláb, 1977). Cold-stage 129 electron microscopy with freeze-fracturing of cheese has been used to examine 130 microstructure (Kaláb, 1979a). Cryo-SEM has allowed examination of food products 131 that contain water, such as cheese, without the need to remove the water phase, 132 thus minimising alteration of the structure during the sample preparation stage.

133 Recent developments in SEM technology, in particular field emission electron optics 134 and environmental specimen chambers, will allow high resolution studies in the 135 future of cheese microstructure at atmospheric pressure without the need for 136 extensive sample preparation. 137 Scanning tunnelling microscopy (STM) offers the advantage of high resolution 138 with minimal impact upon the material being examined from sample preparation, 139 however it requires a specimen that is electrically conducting (Binnig & Rohrer, 140 1982). Atomic force microscopy, a descendent of STM, measures the force on a 141 probe or its deflection as it moves along the surface of a biological sample, thus 142 obviating the conductivity requirement (Binnig et al., 1986).

The resolutions of 143 different types of microscopes are shown in Fig. 1 together with the size of 144 constituents of milk. 145 146 2.2. Differential scanning calorimetry 147 148 Differential scanning calorimetry has been used to differentiate between 149 natural and imitation Mozzarella cheese made from calcium caseinate, through the 150 proposed mechanism of altering the crystallisation properties of the milk fat in 151 cheese (Tunick et al., 1988). Changes in fat crystallisation warrants further 152 investigation as it will certainly impact upon fat globule structure and functional 153 properties such as free oil formation (see sections 3.3.2 and 4.4).

This technique 154 may also find potential use in identifying the mammalian source of milk used in 155 cheese manufacture (Karray et al., 2004), or in examining the differences in cheese 156 melting profile (Famelart et al., 2002). 157 158 2.3. Magnetic resonance imaging and nuclear magnetic resonance 159 160 Magnetic resonance imaging has proved useful for examining the gross 161 microstructure of cheese, including the location of holes and slits within the bulk of 162 the cheese block (Rosenberg et al., 1992), thus providing a non-invasive method for 163 cheese quality control. This technique has also been used to measure moisture and 164 fat content in cheese (Ruan et al., 1998; Budiman et al., 2002), and to assess 165 cheese ripening by quantifying a selection of chemical compounds in Parmigiano 166 Reggiano cheese using high resolution magnetic spinning nuclear magnetic 167

5 resonance spectroscopy (Shintu & Caldarelli, 2005). 168 Nuclear magnetic resonance (NMR) has been employed to measure water 169 mobility in Mozzarella cheese, providing evidence for at least two phases of water, 170 the more mobile bulk water trapped within the casein matrix and a less mobile 171 water phase bound directly to the casein (Kuo et al., 2001). This technique has also 172 been used to examine the effect of freezing of Mozzarella cheese where ice crystals 173 open up the protein matrix and allow development of water pockets (Kuo et al., 174 2003). Pulsed field gradient NMR can be used to assess the diffusion of water 175 molecules and fat globules in cheese (Callaghan et al., 1983).

This technique has 176 estimated the size of serum pockets to be around 10 µm and the size of fat globules 177 to be around 2-3 µm, consistent with microscopic observations. 178 179 2.4. Dynamic rheology 180 181 Dynamic oscillatory rheometry offers a method to indirectly probe the structure 182 of cheese over a range of experimental time scales, extents of deformation, and 183 temperatures. Small amplitude oscillations are imposed upon the cheese sample, 184 such that the strains are within the linear viscoelastic region where any structural 185 breakdown is largely reversible within the time-scale of the experiment.

Exceeding 186 this limit causes the cheese structure to change and re-form into a different 187 conformation upon cessation of the oscillatory strain. 188 The experimental time scale is defined as the reciprocal of the oscillation 189 frequency. Large time scale experiments (at slow frequency) allow sufficient time for 190 flow units within a sample to move and rearrange during the experiment, thus the 191 sample is more fluid-like. Short time scale experiments do not allow sufficient time 192 for the flow units to move, thus the sample is more solid-like. Given enough time all 193 materials flow to some extent, as noted by the Hebrew prophetess Deborah who 194 observed that the "mountains flowed before the Lord" (Judges 5:5, c.

1229 BC), 195 thus neatly bringing together the two disciplines of theology and rheology. The 196 Deborah number is a measure of flowability, and is equal to the ratio of the inherent 197 relaxation time of a material to the experimental time scale. Obviously mountains do 198 not flow particularly well, so these will have exceedingly large Deborah numbers. 199 One consequence of the Deborah number is that the elasticity will depend upon 200 how fast the experiment is performed, in other words, the higher the frequency of 201 oscillation, the more likely the material will be solid-like. The degree of solidness or 202 fluidity is quantified by the loss-tangent value (tan δ), equal to the inverse tangent of 203 the ratio of the elastic loss modulus (G") to the elastic storage modulus (G').

Both 204 elastic moduli are calculated from the ratio of stress to strain, so for a given applied 205 stress, any factor that reduces the strain will increase the elasticity and firmness. 206 Delta (δ) is the phase angle between stress and strain in an oscillatory experiment. 207 For low values of δ towards zero degrees, the material is more solid or gel-like, 208 whereas higher values towards 90° indicate a more fluid-like substance. A more 209 detailed description of rheological test methods is provided by Sherman (1970) and 210

6 van Vliet et al. (1991). 211 212 2.5. Dynamic light scattering 213 214 The size of colloidal components in milk, such as casein micelles and the 215 smaller size population of fat globules, can be determined by fluctuations in the 216 intensity of scattered light using the technique of dynamic light scattering (Dalgleish 217 & Hallet, 1995). The extent of interaction between long-chain molecules, such as 218 proteins and polysaccharides, and colloidal components in milk can thus be 219 examined. This techniques is particularly useful for probing changes in 220 hydrodynamic diameter that may occur after adsorption of proteins onto the 221 surfaces of small fat globules, thus providing evidence that caseins interact with the 222 surface of fat globules and hold them into place in the cheese matrix (Su & Everett, 223 2003).

A schematic diagram of this type of measurement is shown in Fig. 2. 224 225 3. Processing effects 226 227 3.1. High pressure processing of milk 228 229 High pressure (600 MPa) treatment of skim milk results in a cheese with a 230 dense network of casein strands, as observed by SEM, formed from partially 231 disintegrated casein micelles (Needs et al., 2000). As a result, G' is higher (due to a 232 greater density of casein chain cross-links), and syneresis is reduced due to the 233 retention of whey in micro-pockets of serum phase. High pressure treatment of milk 234 increases the rate of firming of renneted milk (Hayes & Kelly, 2003), consistent with 235 a greater casein cross-link density, and with an increased gel strength (Huppertz et 236 al., 2005).

Treatment of milk at high pressures of around 500 MPa reduces the size 237 of milk fat globules and disperses these more evenly in a more compact protein 238 matrix, producing a cheese that is firmer and more elastic (Buffa et al., 2001). The 239 effect of high pressure treatment of milk is to produce a cheese that flows more 240 readily, at least in the case of young Gouda (Messens et al., 2000). High pressure 241 treatment of goats' milk prior to cheese manufacture produces thinner strands of 242 casein with more moisture retention in the final cheese (Guerzoni et al., 1999). The 243 calcium mineral balance and milk fat crystallisation are both affected by high 244 pressure (Huppertz et al., 2002).

245 As well as pressurisation of milk, cheese can also be treated by high pressure. 246 Pressure treatment of immature Mozzarella cheese increased meltability and 247 hardness (Johnston & Darcy, 2000), and increased the swelling of the casein matrix 248 concomitant with greater water retention (O'Reilly et al., 2002). Shredding of freshly 249 made Cheddar cheese is enhanced by pressurisation (Serrano et al., 2004). It is 250 clear that high pressure processing of milk or cheese can provide an opportunity to 251 influence the functional properties of cheese. 252

7 3.2. Homogenization of milk 253 254 Homogenization of cheese-milk creates smaller globules with a greater total 255 fat-water interfacial surface area. To prevent immediate globule coalescence the 256 newly created interface must be coated with surface-active components present in 257 the serum phase, usually casein micelles, micellar fragments and whey proteins. 258 The casein component of the new interfacial area is capable of interacting with the 259 surrounding casein matrix to participate in the structure. The outcome is a firmer 260 cheese with less free oil formation (Rowney et al., 2003a) compared to non- 261 homogenised cheese with the same fat content.

Homogenization of milk prior to the 262 manufacture of cream cheese allows the fat globules to interact with the casein 263 matrix and produce a more elastic cheese with less free oil formation. Microscopy, 264 such as TEM and cryo-SEM, has shown that larger fat globules and less adsorption 265 of casein micelles onto the globule surface enhance spreadability of American-style 266 cream cheese containing a minimum of 33% fat (Kaláb, 1985). 267 The effect of homogenization of cheese-milk is to increase the whiteness of 268 cheese by reducing the size of globules such that light scattering is more effective, 269 and also to increase moisture retention in the finished cheese (Rudan et al., 1998).

270 Reduced-fat Mozzarella cheese is less white due to a smaller number of colloidal 271 sized fat globules able to scatter light, and more translucent due to less fat that 272 would otherwise impair transmission of light (Rudan et al., 1999). Opacity of 273 Mozzarella cheese is a thermoreversible process; heating the cheese increases the 274 whiteness level due to scattering by serum pockets and protein aggregates that are 275 of similar size to the wavelength of light (Pastorino et al., 2002; Joshi et al., 2003). 276 Cooling the cheese gives a translucent appearance as light scattering intensity is 277 reduced.

This reversibility may be due to proteins aggregating and contracting at 278 higher temperatures due to stronger hydrophobic association. 279 Microfluidization is a process where streams of a coarse emulsion collide 280 under high pressure to create a very fine emulsion through turbulence and 281 cavitation. Milk that has been Microfluidized contains casein micelles with very 282 small embedded fat globules, indicating that this high pressure homogenisation 283 disrupts the casein micelle structure (Dalgleish et al., 1996). This may be employed 284 to create different functionality in cheese made from Microfluidized milk.

285 286 3.3. Pasta filata process 287 288 Some varieties of cheese, most notably Mozzarella, are manufactured for 289 functional properties rather than for flavour. These types of cheese are commonly 290 used by the food service sector as ingredients. Low-moisture Mozzarella cheese is 291 manufactured as a cheese ingredient, and is commonly known as pizza cheese. The 292 freshly-made curd is stretched under hot water (typically between 55° and 80°C) in 293 a pasta filata (stretched curd) process to impart desirable stretch and melting 294 characteristics. The elongation of protein fibres and aggregated fat globules and 295

8 pools of fat in the direction of stretching can be seen by dual-labelled CLSM 296 (Rowney et al., 2004); (Fig. 3). 297 Heating and stretching freshly made Mozzarella curd stretches the casein 298 fibres in the direction of elongation with water channels located between the fibres 299 (McMahon et al., 1993). This has been observed using electron and visible light 300 microscopy (Taneya et al., 1992). The water channels are also stretched during this 301 process (Poduval & Mistry, 1999; Joshi et al., 2004). Salting of the curd enhances 302 protein swelling and hydration, which results in bulk water being absorbed by the 303 casein phase.

As a consequence, the amount of expressible serum by 304 centrifugation is reduced within the first 10-20 days after manufacture (McMahon et 305 al., 1999; Guinee et al., 2002; Everett et al., 2004). Fat globules (Guinee et al., 1999) 306 and bacterial cells are located within the water channels, and these are compressed 307 during protein swelling. Indentations of fat globules and cells are observed by SEM 308 on the interior surface of the water channels (McMahon et al., 1999). 309 Casein hydration is enhanced by high levels of sodium chloride and low levels 310 of ionic calcium, and is manifested as an expansion of the casein matrix in cheese.

311 Sodium chloride enhances, whereas calcium ions decrease casein micelle solvation 312 (Creamer, 1985). Increased hydration leads to enhanced casein-water interactions 313 with reduced casein-casein interactions, and increased meltability as casein 314 aggregates flow more easily (McMahon & Oberg, 1999; Joshi et al., 2004). Depletion 315 of calcium in Mozzarella cheese one day after manufacture causes swelling of the 316 casein matrix, indicating protein hydration is enhanced (Guinee et al., 2002). Casein 317 hydration may also facilitate increased proteolysis (Joshi et al., 2003). 318 319 3.3.1.

Cheese melting 320 321 Cheese melting can be assessed by measuring the increase in the diameter to 322 height ratio of cheese cores at different temperatures (Arnott and Schreiber tests), 323 or flow in a heated tube (Olson & Price, 1958). More recent instrumental techniques 324 include dynamic rheology (Guinee et al., 1999) and squeeze-flow rheometry (Wang 325 et al., 1998). 326 Cheese meltability is improved by enhanced casein solvation, increased 327 temperature, age, proteolysis, fat and moisture levels. Sufficient casein hydration to 328 promote interaction between caseins and the water phase is a requirement for 329 cheese to melt.

Any factor that decreases casein-casein interactions will increase 330 cheese meltability. These include reducing the pH to around 5.2, maintaining a 331 higher ratio of soluble to insoluble calcium, and reducing total calcium by chelation, 332 washing of the curd, pre-acidification of milk, or draining the curd at a lower pH. 333 Directly acidifying cheese milk rather than culturing will decrease the amount of 334 bound calcium at the same pH. Mozzarella cheese melts poorly below pH 5 due to 335 incomplete fusion of casein aggregates (Kindstedt et al., 2001). The effect on 336 meltability and the proportion of soluble calcium is reversible over the pH range of 337 4.8 to 6.5 (Ge et al., 2002).

Although pH is known to affect meltability, this may be 338

9 through the mechanism of altering the ratio of bound to soluble calcium. Total 339 calcium is probably not as important as the bound calcium to casein ratio in 340 dictating melting characteristics (Lucey et al., 2003). 341 Casein solvation improves the meltability of Mozzarella cheese by enhancing 342 casein-water interactions. This interaction increases as the cheese ages due to a 343 transfer of water from the bulk phase to the entrapped phase. The casein phase has 344 been argued to be more important than the fat in dictating melting behaviour 345 (McMahon & Oberg, 1998; Guinee et al., 1999).

346 Other factors that increase meltability include greater proteolysis (longer 347 ripening time), higher levels of fat and water (Tunick et al., 1993), using emulsifying 348 salts in the case of process cheese, and increasing the level of free oil (McMahon et 349 al., 1999) which perhaps provides a lubricating role. Concentrating milk prior to 350 cheese manufacture, or homogenising cheese milk will have an adverse effect on 351 cheese melting. Fat globules coalesce in cheese above about 60°C, as observed by 352 CLSM, and this coincides with an increase in the value of tan δ from dynamic 353 rheological measurements, concomitant with increased fluidity (Auty et al., 1999).

354 355 3.3.2. Free oil formation 356 357 The manufacture of pizza-style cheeses requires the application of heating and 358 stretching, a process that is likely to exacerbate unwanted formation of free oil 359 when the cheese is heated on a pizza base. Although a certain amount of free oil is 360 necessary to prevent localized dehydration of the surface of cheese blisters, with 361 associated browning of the surface (Rudan & Barbano, 1998), large amounts of oil 362 on the cheese surface are unappealing to the pizza aficionado. 363 A study by Rowney et al. (2003) examined the effects of curd deformation and 364 temperature on free oil formation.

Stretching curd to a greater extent in the pasta 365 filata process increases the propensity for free oil forming, whereas increasing the 366 rate of stretching has a much smaller effect on free oil. Compression of curd has 367 little effect. Higher temperatures increase free oil, perhaps by promoting fat globule 368 coalescence and rupture. 369 In freshly made one-day old Mozzarella cheese, free oil formation in the heated 370 cheese is not affected by the level of salt (Rowney et al., 2004), however free oil is 371 reduced at higher salt levels in cheese that is further ripened (Everett et al., 2004).

372 The expanded volume of water channels in fresh curd contains fat globules that 373 apparently are under little pressure to deform by the surrounding casein matrix. As 374 cheese ages and the water channels shrink, a process that is hastened by higher 375 salt levels, the fat globules begin to be compressed by the protein matrix. At higher 376 salt concentrations fat globules embed into the adjacent casein matrix, thus provide 377 a protective barrier to rupture, with less free oil. Without this process of salt-induced 378 casein expansion occurring, fat globules are more easily ruptured to produce free 379 oil by a mechanism that may involve aggregation, coalescence and proteolysis of 380 the fat globule membrane layer.


10 In conjunction with the salt-induced swelling of the casein matrix, the apparent 382 viscosity of Mozzarella cheese increases at higher salt levels due to partially 383 inhibited proteolysis (Everett et al., 2004). This might be expected to increase the 384 propensity for fat globules to rupture as the casein matrix expands, however this is 385 not the case. The increase in viscosity is a much smaller effect than the swelling of 386 the casein matrix during the early stages of ripening, thus globules are protected 387 against rupture by embedding into the soft casein fibres. 388 389 3.4.

Freezing of cheese 390 391 Slow freezing of cheese produces large ice crystals, whereas rapid freezing 392 produces smaller crystals. Large crystals of ice can disrupt the cheese protein 393 matrix, as shown by SEM, and increase the meltability of Mozzarella cheese (Oberg 394 et al., 1992). Large ice crystals can create serum pockets within the protein matrix, 395 as shown by the increase in water mobility by NMR measurements (Kuo et al., 396 2003). Frozen Mozzarella cheese has a more porous protein structure, as shown by 397 SEM, due to ice crystal formation (Graiver et al., 2004). The effect on frozen and 398 thawed non-pasta filata cheese is similar to non-frozen pasta filata cheese, in that 399 both contain large pockets of trapped water.

If the freezing rate is sufficiently fast to 400 produce small crystals, the impact upon protein matrix disruption may be minimal 401 (Cervantes et al., 1983). Freezing has also been shown to decrease ewes’ milk 402 cheese firmness, presumably by reducing the density of casein cross-linkages and 403 creating a more porous cheese (Fontecha et al., 1994), and quantified by a smaller 404 value of G'. 405 406 4. Composition of cheese 407 408 Cheese is a complex array of components such as fat globules, pools of 409 entrapped free fat, bacterial and yeast cells, and minerals contained within a casein 410 network interspersed with serum channels.

The casein network is made up of 411 micelles from milk, however these fuse together to form chains during the early 412 stages of cheese manufacture and eventually lose their discrete structure (Kimber et 413 al., 1974; de Jong, 1978). The micelles contain insoluble colloidal calcium 414 phosphate (CCP) that binds casein molecules through interaction with 415 phosphorylated serine amino acids. All of these structural components, in addition 416 to components such as soluble minerals and organic acids which don't contribute 417 directly to structure, contribute to the flavour profile of cheese.

It has been noted 418 that cheese with poor texture has poor flavour (Lawrence et al., 1983), although it is 419 not necessarily the case that good textured cheese has good flavour. This 420 underscores the importance of achieving a correct balance of the structure 421 components to produce an acceptable cheese. 422 The rheological properties, as a method to quantify texture, depend upon the 423

11 relative amounts and extents of interaction between cheese structural units. The 424 factors that impact upon texture include fat globules occluded within the protein 425 matrix, fat globules coated with casein micelle fragments (as a consequence of 426 homogenisation) that interact with the surrounding casein matrix, free pools of fat, 427 casein matrix density, proteolysis (which reduces the casein cross-link density), 428 water content, and the bond strength and density between chains of fused casein 429 micelles. Increasing the casein bond strength and the density of bonds will increase 430 the firmness of the matrix.

431 Cheese ripening has a profound affect on texture and structure. In Mozzarella 432 cheese fat globules aggregate and coalesce over time, creating large fat particles 433 that perhaps contribute to free oil formation. Larger globules of fat require a lower 434 Laplace pressure to induce rupture, thus are more likely to contribute to free oil. 435 Proteolysis from residual chymosin, indigenous milk enzymes such as plasmin, and 436 proteases from starter bacterial cells and other microorganisms undoubtedly 437 contribute to a softening of cheese. Casein hydration occurs over time, particularly 438 in pasta filata varieties, contributing to increased cheese melting characteristics and 439 a reduction in expressible serum.

Calcium equilibria is known to impact 440 considerably upon cheese firmness (O'Mahony et al., 2005). Over time, calcium 441 bound to the casein matrix solubilises, thus lessening inter-casein molecular 442 interactions (see section 4.2). 443 Cheese softening over time can be simplified as a two-step process; the slow 444 solubilisation of bound calcium, and proteolysis. Small peptides in the serum phase 445 play a lesser structural role than intact caseins so the increase in proteolysis during 446 cheese ripening will soften cheese. A reduction in bound calcium will reduce 447 electrostatic interactions between casein and also soften cheese.

The long time 448 required for solubilisation of calcium salts can perhaps be explained by noting that 449 a saturated calcium ionic solution surrounding the insoluble calcium phosphate 450 within the casein aggregate structure will hinder further dissolution. As calcium ions 451 slowly diffuse out of the adjacent micro-serum pockets and away from the insoluble 452 aggregates, the solubilisation process can further continue. In addition, the increase 453 in HPO3 2- and H2PO3 - from the dissolving colloidal calcium phosphate bound to 454 casein will increase the buffering capacity of the serum phase surrounding the 455 calcium phosphate aggregates, further slowing the rate of solubilisation.

456 457 4.1. Water 458 459 Water exists in three main phases in cheese: bulk water in the serum channels, 460 entrapped water in close proximity to the casein matrix, or as bound water tightly 461 associated adsorbed to the caseins and therefore unavailable as a solvent. Heating 462 and stretching fresh curd increases the proportion of mobile water molecules in 463 Mozzarella cheese (Kuo et al., 2003), and this is thought to represent the bulk water 464 phase. In the first few weeks of ripening the proportion of entrapped water 465 increases at the expense of bulk water, brought about by casein swelling and 466

12 hydration (McMahon et al., 1999). This is confirmed by water mobility 467 measurements using NMR (Chaland et al., 2000; Kuo et al., 2001). Water mobility 468 measurements using NMR have shown that the protein phase in Mozzarella is less 469 hydrated than in Gouda, due to the pockets of serum phase in the former, and the 470 degree of casein hydration increases with age (Godefroy et al., 2003). Non-pasta 471 filata cheese has differing relative amounts of the three states of water, as 472 measured by NMR, compared to Mozzarella cheese. The amount of bound water is 473 related to the protein content, however an unexpected relationship has also been 474 observed between fat and bulk water (McMahon et al., 1999).

This can be explained 475 by the SEM observation that fat globules are primarily located in these water 476 channels in fresh Mozzarella cheese. 477 478 4.2. Calcium 479 480 The effects of calcium addition to milk and pH are not independent events. 481 Increasing Ca2+ also reduces pH due to chelation of HPO3 2- and H2PO3 - ions, thus 482 the increase in milk gel firmness and the reduced time for clotting to occur may be 483 due to a decrease in pH rather than an increase in soluble calcium. 484 A reduction in the ratio of insoluble to soluble calcium is known to increase 485 cheese meltability.

This reduction can be facilitated by the addition of citrate, which 486 chelates calcium ions, or by decreasing the pH. Examination of Cheddar cheese by 487 SEM has shown that cheese with added citrate contains an expanded protein 488 matrix (Pastorino et al., 2003c), concomitant with the idea that a decrease in casein- 489 bound calcium reduces casein-casein interactions, thus facilitating flow during 490 melting. 491 As the pH of cheese is reduced towards the iso-electric point of casein (pH 492 4.6), there is a contraction of the casein matrix brought about by an increasing 493 propensity for hydrophobic association of micellar aggregates.

The size of protein 494 sub-aggregates is reduced from 10 nm to 2 nm as pH decreases from 5.3 to 4.7 495 (Pastorino et al., 2003a), illustrating well the compaction and rearrangement of the 496 micellar structure in fresh curd as the pH is reduced over time. At a pH of around 497 5.6 the casein structural units interact through calcium-mediated electrostatic 498 bonds. As pH is further lowered, the bound calcium solubilises, resulting in 499 interactions between casein units by increasingly hydrophobic association. A 500 maximum in flowability occurs at around pH 5.2 to 5.4. At higher pH values where 501 calcium-mediated interactions are still predominant, this reduction in casein sub- 502 aggregate size is correlated with an increase in cheese flowability (Lawrence et al., 503 1983).

504 Increasing the amount of total (ionic plus insoluble) calcium in cheese 505 enhances casein-casein interactions, thereby reducing meltability and increasing 506 cheese firmness (Joshi et al., 2003). A consequence of enhancing casein 507 interactions through insoluble (not soluble) calcium is the closing up of serum 508 pockets, with less expressible serum. This is a consequence of caseins aggregating 509

13 via bound calcium phosphate. Increasing pH above 5.4 reduces meltability, 510 however if the amount of calcium is reduced at this higher pH, protein hydration and 511 swelling is promoted, concomitant with improved melting and stretching behaviour 512 (Guinee et al., 2002). 513 514 4.3. Sodium chloride 515 516 Sodium chloride (salt) is added to freshly made curd to limit the action of 517 starter bacteria from reducing the pH to unacceptably low values, and assisting in 518 whey expulsion immediately after curd milling (in the case of dry-salted cheeses), as 519 well as the secondary effect of flavour enhancement.

A review of the effects of salt 520 in cheese has been provided by Guinee (2004). Higher levels of salt induce a 521 swelling of the casein phase with subsequent adsorption and absorption of 522 moisture by the casein matrix, up to at least 1.6% NaCl in Mozzarella, as shown by 523 Rowney et al., (2004). This has been observed with Mozzarella cheese and with 524 Muenster cheese (Pastorino et al., 2003b). The protein phase in unsalted cheese will 525 swell at a slower rate compared to salted cheese, and this explains the slower rate 526 of reduction of expressible serum over time (Guo et al., 1997).

Such unsalted 527 cheese has larger pockets of serum phase. Unsalted directly acidified Mozzarella 528 cheese contains a more porous protein matrix as a consequence of enhanced 529 casein-casein interactions, thus impaired meltability (Paulson et al., 1998). 530 The application of salt will draw moisture out of a curd particle. Paradoxically, 531 if this is done over a short period of time the water will remain entrapped in the curd 532 structure. This is due to the formation of a cheese surface barrier that impedes the 533 flow of water. The movement of water from Ragusano, a Sicilian brine-salted 534 cheese, is impeded due to the formation of a dense dehydrated protein layer at the 535 surface of the cheese, as observed by SEM (Melilli et al., 2003).

This effect is 536 enhanced by higher salt levels and longer brining time (Melilli et al., 2005). 537 538 4.4. Fat globule microstructure 539 540 Fat is found in cheese either as small globules of order 2 µm in size, 541 aggregates of globules, or as large areas of fat 10-50 µm in size that are most likely 542 pools of free fat trapped within the protein matrix (Everett et al., 2003). Analysis of 543 the size and shape of fat areas in cheese can be done by computerized 2-D and 3- 544 D analysis of confocal micrographs with appropriate fluorescent staining of the fat 545 or the aqueous protein phase (Everett et al., 1995).

Aggregates of fat globules in 546 Cheddar cheese are seen to align along the direction of the casein fibres (Hall & 547 Creamer, 1972), presumably due to the forces imposed by the cheddaring process. 548 Elongation of fat globules is not observed in Gouda and Edam, where cheddaring or 549 stretching do not take place during manufacture (Kaláb, 1977), or in Cheshire (Hall 550 et al., 1972). 551

14 Fat globules physically interfere with the integrity of the cheese casein matrix 552 and soften texture. This process will occur if the globules act as inert filler material, 553 or to a lesser extent if the globules participate as copolymers with the casein matrix, 554 as will be the case when globules are homogenized and partially coated with casein 555 micelle fragments (van Vliet & Dentener-Kikkert, 1982; Cano-Ruiz & Richter, 1997). 556 There is some debate about whether natural fat globules are inert filler material or if 557 there is some interaction with the surrounding casein matrix.

The observation using 558 cryo-SEM that fat globules are surrounded by serum cavities in fresh Cheddar 559 cheese supports the idea of globules being inert filler material (Hassan & Awad, 560 2005). Cheddar cheese containing fat globules coated with either the non-ionic 561 surfactant Tween 80 or with the zwitterionic phosphatidylcholine were observed to 562 have a high loss of fat after cutting the curd (one-third of fat was lost with Tween 563 80-coated fat globules), in contrast to natural fat globules where more than 90% (by 564 volume) were retained within the curd (unpublished results, D.W.

Everett). This 565 suggests that natural fat globule are held into place within the casein matrix by 566 some interaction with the native globule membrane coating. A comparison between 567 cheese confocal micrographs with native fat globules and globules coated with 568 phosphatidylcholine is shown in Fig. 4, showing a lesser amount of fat in cheese 569 containing globules coated with phosphatidylcholine. In both cases, fat globule size 570 was standardised to 2 µm in diameter prior to addition to the cheese-milk. Smaller 571 globules are more likely to fit into small voids in the casein matrix and be retained in 572 the curd structure, compared to large globules, in the case where globules are 573 coated with phosphatidylcholine.

For native fat globules, some binding with the 574 casein matrix would prevent the loss of the larger globules. 575 The state of fat is important for dictating the sensory properties of dairy 576 products (Lopez, 2005). Fat globules coated with caseinate and recombined with 577 skim milk for cheese manufacture tend to be more spherical compared to native 578 globules, which are distorted and partially aggregated. Manufacture of cheese 579 containing globules coated by homogenisation with relatively poor emulsifiers such 580 as αs2-casein produce large pools of fat in the cheese that may explain the observed 581 greasy and crumbly texture (Everett et al., 2003).

Evidently pools of fat do not 582 integrate well into the casein matrix. The larger globules produced by 583 homogenisation of milk fat with αs2-casein (compared to other caseins that are 584 better emulsifiers) are apparently easier to rupture, producing oil that is located 585 within voids in the casein matrix and that does not bind to the matrix. In this case, 586 no strengthening of the cheese protein matrix takes place. 587 The structure of fat has implications for the melting of cheese and for free oil 588 formation. Fat globule rupture is likely to lubricate layers of casein and allow easier 589 flow, thus enhancing melting.

Cheese made from recombined milk containing a 590 lower melting point fraction of anhydrous milk fat has more free oil formation and 591 lower viscosity (improved melting), presumably as the more liquid fat phase is able 592 to permeate amongst layers of casein and facilitate melting (Rowney et al., 2003a). 593 Pools of free fat would likely contribute to free oil formation on the surface of heated 594

15 cheese. One application of manufacturing cheese from recombined milk containing 595 fat globules coated with specific emulsifiers is to produce a cheese where the 596 globules have built-in rupturability. The globules can be induced to partially rupture 597 over time to produce a small amount of free oil that may be required for specific 598 cheese functionality, such as enhanced spreadability or meltability. 599 The size of globules will impact upon texture. Larger globules with a smaller 600 Laplace pressure of deformability will decrease the firmness of cheese. Conversely, 601 small globules are much harder to deform and rupture, and are also less likely to 602 disrupt the casein matrix, thus contributing to a firmer cheese.

Confocal microscopy 603 is a simple technique to assess globule size. It is preferable to optically section into 604 the cheese and take a succession of two-dimensional micrographs with subsequent 605 assembly into a three-dimensional structure by computerized image analysis. 606 Although this is a process that requires large computing power, it eliminates the 607 problem of estimating the size of non-spherical globules from two-dimensional 608 micrographs. Although analysis of a two-dimensional image can potentially 609 underestimate the size of a fat particle by taking a section through the tip of the 610 structure, stereology can be used to derive statistically valid data on size and shape 611 from two dimensional images (Langton & Hermansson, 1996).

612 Fat globules aggregation is reduced by homogenisation of milk prior to cheese 613 manufacture, as observed by CLSM (Guinee et al., 2000). Flowability and 614 stretchability of cheese is adversely affected in this case, and is quantified by a 615 reduction in tan δ measured by dynamic rheology. Aggregation is particularly 616 evident when cheese is heated (Paquet & Kaláb, 1988; Auty et al., 1999). In pasta 617 filata cheese varieties the fat globules aggregate—as seen by SEM— (Tunick et al., 618 1993), and coalesce to form pools of free oil within the interstices of the casein 619 matrix.

Extensive fat globule aggregation and rupture is believed to contribute to 620 free oil formation. These pools of free oil have a large degree of distortion (Michalski 621 et al., 2004), as observed by CLSM. One issue with labelling of fat in cheese and 622 observation using CLSM is that there is no clear way to see if elongated fat 623 structures are composed of aggregated spherical globules, or if the entire structure 624 is a pool of free fat trapped within the protein matrix. One solution is to label the 625 cheese with fluorescent tags conjugated to anti-bodies to a component of the fat 626 globule membrane to measure the extent of membrane rupture.

Often these 627 conjugated labels are difficult to produce and expensive to purchase (if available). 628 Process cheese is manufactured by heating natural cheese in the presence of 629 emulsifying salts such as sodium salts of citrate, orthophosphate, pyrophosphate, 630 and sodium aluminium phosphates. The structure differs from natural cheese in that 631 there are no evident curd granules, but there is a more uniform protein matrix and 632 less fat globule aggregation. Higher heating and mixing speeds promote fat globule 633 homogenisation and smaller globule size. Emulsifying salts are added to process 634 cheese to promote the peptisation (solubilisation) of the casein matrix and allow 635 caseins to function effectively as soluble emulsifiers of milk fat that is released 636 during heating of the cheese.

As a result of the addition of these salts, caseins are 637

16 hydrated, calcium ions are chelated and pH rises. All of these mechanisms promote 638 peptisation of the casein matrix. As the degree of emulsification of the fat globule 639 increases, meltability (Savello et al., 1989), free oil formation, and globule size 640 decrease, whereas firmness increases as the globules are able to participate as co- 641 polymers with the surrounding casein matrix. This increase in firmness is in 642 comparison to cheese containing the same fat level, but where the globules are not 643 homogenised. 644 645 4.4.1. Fat substitutes 646 647 There is a push by nutritionists to reduce the amount of saturated fat in cheese 648 for health reasons, however this reduces the flavour and increases the firmness of 649 cheese to levels that may be unacceptable to the consumer.

The use of proteins, 650 carbohydrates, or modified triacylglycerides such as sucrose polyesters to replace 651 fat has found some efficacy. Protein- and carbohydrate-based fat mimetics serve to 652 open up the protein matrix and enhance the formation of water channels, in other 653 words, these mimetics act as casein matrix-breakers (Mackey & Desai, 1995). 654 Larger fat replacers open up larger areas within the casein matrix of Mozzarella 655 cheese, as shown using SEM, and thus are able to increase moisture retention and 656 produce a softer cheese (McMahon et al., 1996). The meltability of Mozzarella 657 cheese can be improved by retaining moisture (Zisu & Shah, 2005).

Maltodextrin 658 and modified potato starch replacers will expand water channels from around 5-10 659 µm to 10-50 µm (Bhaskaracharya & Shah, 2001). The use of larger fat replacers is 660 an effective strategy for opening up the protein matrix and retaining higher amounts 661 of water in Mozzarella cheese (McMahon et al., 1998). This effect has been 662 observed with Cheddar cheese containing a granular lecithin fat replacer (Drake et 663 al., 1998), and would be expected to be more pronounced if the mimetic or replacer 664 is of order micrometers in size. Non-fat Mozzarella cheese without fat replacers 665 contains much lower levels of expressible serum as the near absence of fat 666 globules does not provide for a mechanism to open up the protein matrix to retain 667 bulk water (Paulson et al., 1998).

668 669 4.4.2. Water globules as fat mimetics 670 671 Fat globules serve several purposes in cheese. Firstly, the fat is necessary for 672 flavour development. This can clearly be shown by the uncharacteristic flavour in 673 low-fat cheese (Banks et al., 1989; Jameson, 1990). Whether is this due to fat- 674 soluble flavour precursors, components of the milk fat globule membrane (MFGM), 675 fatty acid flavour precursors, or perhaps the requirement of an oil-water interface for 676 enzymes to maintain high activity and produce flavour compounds is open to 677 debate. In all likelihood, all of these mechanisms play a role in flavour development.

678 It should also be noted that the differences in protein structure between full-fat and 679 non-fat cheese may have some impact upon sensory perception of flavour. 680

17 The second role played by fat globules is a casein matrix structure-breaker, 681 thus reducing the firmness of cheese and providing some lubrication during 682 mastication. A third requirement is to open up the casein matrix to form serum 683 channels. The absence of fat globules yields cheese, in many cases, with a texture 684 that is too firm and with low flavour intensity. 685 Although the continuous phase of cheese is water-based, water globules can 686 be incorporated into skim milk by the addition of polysaccharide stabilisers such as 687 xanthan, pectin, locust bean gum, or guar gum at very low concentrations from 0.01 688 to 0.1%.

The dilute polysaccharide-water phase forms an incompatible system with 689 the polysaccharide-depleted continuous phase, thus forming discrete water 690 globules (Fig. 5). These globules increase in time, however the size can be locked 691 into place by renneting the milk to form a gel (Fig. 6). This open casein matrix 692 structure carries over into the finished cheese (Fig. 7). Current work by the authors 693 is focused on the effect of these water globules on cheese melting. 694 695 4.5. Protein structure 696 697 4.5.1. Milk gelation 698 699 A milk gel can be formed by the addition of rennet at around pH 6.5 as a 700 precursor to cheese manufacture.

Lactic acid bacterial (LAB) cultures are added to 701 reduce the pH. Casein demineralisation occurs as pH decreases, thus reducing 702 electrostatic interactions between casein molecules. Along with this trend, the 703 propensity for hydrophobic interaction of micelles increases as progressive charge 704 neutralisation takes place as pH decreases towards the isoelectric point (Lucey et 705 al., 2003). This latter interaction occurs when sufficient glycomacropeptide has 706 been enzymatically removed from the para-casein by chymosin. These interacting 707 mechanisms can be observed using dynamic rheological methods (Lucey et al., 708 2000).

At high pH around 5.5-6.0, a local maximum of tan δ indicates increased 709 fluidity due to partial demineralisation of the casein micelle, thus less interaction 710 between casein molecules. A local minimum of tan δ at around pH 5.0-5.5 indicates 711 increased solid-like behaviour due to rennet-induced interactions. As pH is further 712 reduced, a transition takes place from a rennet to an acid gel structure. 713 At pH 4.6 rennet gels are more permeable than acid gels, and rennet gels have 714 thicker protein fibres, as established by electron microscopy studies (Roefs et al., 715 1990). As a consequence rennet gels are more able to undergo syneresis, 716 producing lower moisture cheese, compared to acid milk gels.

Rennet gels are 717 more open and porous at pH 6.5 than at pH 5.2 (Hannon et al., 2006). A slower rate 718 of cooling of rennet gels produces a larger number of smaller casein micelle 719 aggregates, more junction zones, and stronger and less permeable gels (Zhong et 720 al., 2004). A higher number density of casein chain cross-links will increase the 721 value of G'. Heating of milk prior to acidification produces a less permeable and 722 firmer acid gel due to casein cross-linking by denatured whey proteins. By contrast, 723

18 unheated acid milk gels are more porous and prone to syneresis, as observed by 724 CLSM and permeability measurements (Lucey et al., 2001). 725 726 4.5.2. Cheese casein network 727 728 A characteristic of cheese ripening is the transformation of a fibrous casein 729 matrix, formed from chains of aggregated micelles, into a more amorphous 730 structure (King & Czulak, 1958; Kimber et al., 1974). Over time the fusion of casein 731 micelle chains creates a more homogenous structure will smaller serum cavities, 732 providing an explanation for the reduction in expressible serum by centrifugation in 733 aged cheese.

During manufacture of cottage cheese, an acid milk gel curd 734 structure, the size of casein micelles increases from around 80 nm to 200 nm 735 (Glaser et al., 1980), consistent with aggregation of micelles into chains during the 736 gelation process. 737 The fusion of curd particles creates junction zones in the ripening cheese. 738 These originate from two aspects of curd fusion. Firstly, thin lines from fused curd 739 particles, and secondly, thicker boundaries deficient in fat globules from fused 740 salted milled curd fingers (Kaláb, 1979b). 741 Cream cheese is manufactured from milk fat standardized to a higher fat 742 content and cultured with lactic acid bacteria to a pH of 4.6.

A defect in some 743 cream cheeses is a gritty mouthfeel. The grittiness may originate from large 744 compacted casein and denatured whey protein aggregates up to 1 mm in size, as 745 seen by SEM (Sainani et al., 2004). 746 The coarseness of the casein matrix can be quantified by TEM. Faster rates of 747 coagulation of UF milk results in a coarser casein matrix with a higher surface area 748 to volume ratio of the casein, and this is positively correlated with a higher stress at 749 fracture in Feta cheese (Wium et al., 2003). Increasing the ratio of caprine to ovine 750 milk in Feta cheese manufacture gave a finer casein matrix, as measured by cryo- 751 SEM, and a firmer cheese (Tsigkros et al., 2003).

Camembert cheese made from 752 reconstituted milk yields a more porous protein matrix due to incomplete fusion of 753 casein aggregates (Peters & Knoop, 1974). An increase in curd acidity, or storage at 754 a higher temperature within a day of manufacture also increases the degree of 755 coarseness of the protein matrix (Knoop & Peters, 1972). 756 757 4.5.3. Casein sub-structural units 758 759 An increase in the size of electron-dense regions from 12 to 23 nm has been 760 observed in Mozzarella cheese over six weeks of ripening using TEM, 761 corresponding to the size of casein sub-structural units (Cooke et al., 1995), and 762 consistent with the idea of increased casein solvation over time.

The spacings 763 between these units also increases during ripening, and perhaps may facilitate 764 increased melting and softening. These units are smaller in size for lower pH 765

19 cheese; 10-15 nm in Gouda cheese at pH 5.3 to 3-4 nm in Cheshire cheese at pH 766 4.6 (Hall et al., 1972), which is correlated with a decrease in elasticity and an 767 increase in brittleness of cheese. 768 769 4.5.4. Proteolysis 770 771 Chymosin has high specificity towards the Phe105 -Met106 bond in κ-casein, 772 however other less specific enzyme preparations, often with higher proteolytic 773 activity, can be used to clot milk and produce cheese curd. Some of these include 774 proteases extracted from microorganisms and plants, and pepsin from a variety of 775 mammals, including bovine sources.

The type of rennet used will impact upon 776 texture, for example, bovine pepsin results in a Cheddar cheese with a more porous 777 structure as seen by SEM, contributing to a softer cheese compared to the use of 778 bovine chymosin (Eino et al., 1976). 779 780 4.6. Mineral crystals 781 782 Calcium phosphate is the most abundant mineral in milk and cheese. Solubility 783 decreases as temperature rises, so this mineral is packaged inside of the casein 784 micelle to ensure it is dispersed in milk. The casein micelle is designed to provide a 785 large amount of calcium to the growing new-born calf in a form that is easily 786 consumed and digested.

Calcium phosphate crystals have been identified in 787 cheese by visible light and electron microscopy, and x-ray microanalysis (Morris et 788 al., 1988). Fluorescence microscopy of natural and process cheese has shown the 789 presence of 10-30 µm crystals (Yiu, 1985). Crystals (20-30 µm in size) have been 790 observed by TEM to be located along seams in cheese, which originate from the 791 lines of curd particle fusion (Brooker et al., 1975). The crystals are perhaps formed 792 from pockets of entrapped whey. Washing of milled curd particles with water or 793 CaCl2 solution removes excess phosphate and prevents the formation of these 794 crystals, identified as CaHPO4.2H20 or brushite (Conochie & Sutherland, 1965).

795 Other methods to observe and identify crystalline structures in cheese include infra- 796 red spectroscopy and energy dissipative x-ray spectrometry analysis. 797 Visible calcium D(-)-lactate crystals (around 80 µm in size) may also form on 798 the surface of aged cheese, which creates an unsightly view to many consumers 799 who may confuse it with mold growth, as well as a crunchy texture to the surface 800 layers. These crystals are formed as a consequence of racemization of L(+)-lactate 801 to D(-)- and L(+)-lactate isomers by non-starter lactic acid bacteria (Johnson et al., 802 1990).

803 804 4.7. Bacterial cells 805 806 Early work has shown that bacterial starter cells preferentially locate near to 807

20 the fat-water interface in cheese (Dean et al., 1959; Kimber et al., 1974). This may 808 have implications for flavour development if the flavour-producing reactions are 809 mediated by enzymes located within these cells, or if there is a requirement for 810 components of the fat globules to act as substrates. Starter bacterial cells are 811 located in the serum channels of Mozzarella cheese, along with fat globules. As the 812 protein phase absorbs and adsorbs water, the serum channels are reduced in size 813 and bacterial cells are observed to be embedded in the swelling casein matrix 814 (McMahon et al., 1999).

Starter bacterial cells are located near to the MFGM layer, 815 and the number of cells is related to the fat content of cheese (Laloy et al., 1996). 816 Probiotic bacteria can be added to milk for cheese manufacture, however the 817 question remains as to the viability of these organisms during cheese ripening. A 818 rapid method using CLSM has been developed to distinguish between live and 819 dead probiotic bacteria (Auty et al., 2001b). This method may give a more accurate 820 estimation of total cell numbers compared to the standard plate count procedure. 821 Exopolysaccharides (EPS) in Feta cheese have been observed using CLSM as 822 thick sheets filling the pores within the casein network (Hassan et al., 2002).

Strains 823 of starter bacteria that produce EPS yield a cheese with larger serum pockets, a 824 less compact protein matrix, and a texture that is less firm (Dabour et al., 2006). 825 826 4.8. Surface flora 827 828 Lactic acid bacteria are added to reduce the pH of milk and to initiate the 829 transformation into cheese. The primary function of LAB is to produce lactic acid 830 from lactose, which reduces the pH from 6.7 for fresh milk to around 5-6 for many 831 varieties of cheese. To add complexity to cheese ripening, some varieties of cheese 832 have a surface layer of bacteria (for example, smear-ripened cheeses such as Tilsit 833 and Brick) or mold (Camembert and Brie), or contain mold in the interior (Roquefort, 834 Gorgonzola, and Stilton).

A complex series of biochemical and physical events take 835 place at the surface from the action of surface microflora. 836 Surface microflora in Camembert and Brie raise the pH by production of 837 ammonia from catabolism of proteins after the supply of lactose at the surface is 838 exhausted. A pH gradient is set up from the surface to the exterior, precipitating 839 calcium phosphate on the surface and creating a second gradient of calcium ions, 840 which migrate to the surface, leaving a softer texture in the interior. The increase in 841 pH in the cheese during ripening facilitates the action of plasmin, contributing 842 further to cheese softening.

Thus this type of cheese is said to ripen from the 843 outside in (Noomen, 1983; Karahadian & Lindsay, 1987; see Fig. 8). 844 845 5. Future developments 846 847 It is a condicio sine qua non that dairy industry profits will drive food structural 848 research. As such, knowledge that reduces time and energy in cheese manufacture 849

21 or that confers additional health benefits will more likely be funded. These will 850 include reducing the length of time required for cheddaring, reducing ripening time, 851 replacement of the pasta filata process with something requiring less energy, 852 predicting the onset and subsequent cessation of the functional properties of pizza 853 cheese, and fat replacement without detrimental effects on texture and flavour. Milk 854 fat could potentially be replaced with healthier alternatives such as fish oils, utilising 855 encapsulation to contain unwanted tastes and odours. 856 With the continual discovery of novel food ingredients that confer functional 857 properties to food products, it is of both scientific and commercial interest to 858 investigate how these ingredients interact with dairy components in cheese, 859 perhaps using new techniques such as atomic force microscopy under native 860 cheese conditions.

The current interest in polysaccharides offers an opportunity to 861 produce cheese with well defined functional properties (for example, shredding, 862 slicing, melting, stretching) without the need for ripening. Milk fat is considered to 863 be unhealthy so there remains great opportunity to replace fat globules with 864 protein- or carbohydrate-based replacers. One novel approach is to utilise the 865 thermodynamic incompatibility of mixtures of polysaccharide solutions and casein 866 micelle dispersions to form water globules. Knowledge of the textural aspects of fat 867 replacers is currently more advanced than the impact upon flavour chemistry, 868 although this will certainly need to be rectified before complete replacement of fat 869 globules can be achieved.

870 There is a wealth of knowledge on how milk composition and renneting 871 conditions affect gel structure, however the extrapolation of this to cheese structure 872 is largely empirical and less mechanistic details are known. Cheese ripening is an 873 immensely complex and chaotic phenomenon from a structural perspective, to say 874 nothing about the cascade of flavour-producing reactions. The advent of faster 875 computer modelling and a more basic understanding of mineral equilibria, casein- 876 casein and casein-fat molecular interactions will go some way to providing a basis 877 for predicting cheese structure and ultimately texture from the fundamentals of milk 878 coagulation.

879 Perhaps cheese structural work does not have the panache of research into 880 the health benefits of foods. Do we really need to know how different components 881 interact to produce texture? And will this add value to the dairy industry? The rapid 882 increase in pizza cheese production since the 1980s would seem to support these 883 notions. This type of cheese is manufactured for its functional properties, rather 884 than for flavour, so a substantial investment has been made in determining 885 functionality. This is clearly evident when one considers the large increase in the 886 number of published papers on Mozzarella cheese since the early 1990s, research 887 that is conducted in public institutions and funded by the dairy industry, and that 888 apparently is continuing unabated.

Pizza cheese has acceptable functional 889 properties for a short window of opportunity between about 10 days and 7 weeks 890 after manufacture, extending out to as much as six months for partially acidified and 891 cultured Mozzarella. Understanding basic elements of structure will allow us to 892

22 predict when this cheese loses functionality, and more importantly, extending this 893 functionality window. There will be other cheese varieties and other functional 894 issues in the future that will attract industry support. 895 Much of the early work on cheese structure has been conducted by various 896 microscopic techniques. There remains a need to quantify this information such that 897 it can be part of a model that ties together microscopic observations, sensory 898 properties, rheological measurements, and functional properties. The increase in 899 complexity of microscopes (for example, TEM and SEM in the early years, to cryo- 900 SEM, and environmental SEM today) gives us much detail about structure under 901 conditions that more closely approximate that of food products.

Sensory and 902 functional properties are important parts of a future model, particularly as these 903 properties generate income for the dairy industry. More and more sophisticated 904 instrumentation does not come cheaply, but it does provide an opportunity to 905 understand structure at basic molecular level and allow us to make predictions 906 about structural development over the shelf-life of a food product. More importantly 907 it will provide a basis to develop new food ingredients to add value to milk 908 products. 909 910

23 List of Figures 911 912 Figure 1: Resolution of different types of microscopy and the size of milk 913 constituents. 914 915 Figure 2: Measurement of hydrodynamic diameter of a milk fat globule membrane 916 (MFGM) coated colloidal particle using dynamic light scattering. Schematic diagram 917 shows the reduction in diameter after hydrolysis of an adsorbed protein layer at the 918 surface. 919 920 Figure 3: Confocal micrograph of Mozzarella cheese showing the protein phase as 921 red, the fat phase as blue, and the serum phase as black. Scale bar 25 µm. 922 923 Figure 4: Confocal micrographs of Cheddar-like cheese containing, (a) native fat 924 globules, and fat globules coated with (b) phosphatidylcholine, showing less fat 925 retention in the cheese curd.

Black areas are fat globules. Scale bar 20 µm. 926 927 Figure 5: Visible light micrographs showing incompatible phase separation of 5.2% 928 (w/v) non-fat phosphocasein dispersion at pH 7.1 and the formation of water 929 globules containing soluble guar; (a) 0.06%, (b) 0.08%, (c) 0.10%, and (d) 0.12% 930 guar (guar solution inverting to becoming the continuous phase). Micrograph taken 931 30 minutes after mixing. Scale bar 10 µm. 932 933 Figure 6: Confocal microscopy of renneted non-fat 5.2% (w/v) phosphocasein 934 dispersion at pH 6.4, (a, b) in the absence of gum, and (c, d) with added 0.08% 935 (w/v) guar gum.

Micrographs are shown before gelation (a, c) and after the point of 936 gelation (b, d). White is the protein phase; black is the serum phase. Scale bar 25 937 µm. 938 939 Figure 7: Confocal micrographs of salted non-fat Cheddar-like cheese containing, 940 (a) 0.3% pectin, (b) 0.15 % locust bean gum, (c) 0.1% xanthan gum, and (d) control. 941 Micrographs were taken 2 d after manufacture and storage at 4°C. White is the 942 protein phase; black is the serum phase. Scale bar 25 µm. 943 944 Figure 8: Cascade of physical and chemical reactions during ripening of Camembert 945 and Brie cheese. 946 947 948 949

24 References 950 951 Auty M. A. E., Fenelon, M. A., Guinee, T. P., Mullins, C., & Mulvihill, D. M. (1999). 952 Dynamic confocal scanning laser microscopy methods for studying milk protein 953 gelation and cheese melting. Scanning, 21, 299-304. 954 Auty M. A. E., Twomey, M., Guinee, T. P., & Mulvihill, D. M. (2001a). Development 955 and application of confocal scanning laser microscopy methods for studying the 956 distribution of fat and protein in selected dairy products. Journal of Dairy 957 Research, 68, 417-427. 958 Auty M. A., Gardiner, G. E., McBrearty, S. J., O'Sullivan, E. O., Mulvihill, D.

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33 Figure 1 1324 1325 1326 1327 1328

34 Figure 2 1329 1330 1331 1332 1333 1334

35 Figure 3 1335 1336 1337 1338 1339

36 Figure 4a 1340 1341 1342 1343 Figure 4b 1344 1345 1346 1347 1348

37 Figure 5a 1349 1350 1351 1352 Figure 5b 1353 1354 1355 1356 1357

38 Figure 5c 1358 1359 1360 1361 Figure 5d 1362 1363 1364 1365 1366

39 Figure 6a 1367 1368 1369 1370 1371 Figure 6b 1372 1373 1374 1375 1376

40 Figure 6c 1377 1378 1379 1380 1381 Figure 6d 1382 1383 1384 1385 1386

41 Figure 7a 1387 1388 1389 1390 1391

42 Figure 7b 1392 1393 1394 1395

43 Figure 7c 1396 1397 1398 1399 1400

44 Figure 7d 1401 1402 1403 1404 1405

45 Figure 8 1406 1407 1408