Technology of Cheesemaking

1 The Quality of Milk for Cheese Manufacture

T.P. Guinee and B. O’Brien

1.1 Introduction

World production of milk in 2008 is estimated at ~576 × 10⁶ tonnes (ZMP, 2008), with India/Pakistan, the Americas and Europe being the major producing regions. The proportions of total milk produced by cow, water buffalo, goat, ewe, camel and other are ~84.0, 12.1, 2.0, 1.3, 0.2 and 0.2, respectively (International Dairy Federation – IDF, 2008). Cows’ milk is the major milk used for cheese manufacture; however, significant quantities of cheese are also made from goat, sheep and water buffalo milks in some European Union (EU) countries, such as France, Italy and Spain.

Based on an estimated yield of 1 kg cheese 10 kg−1 milk, the percentage of total milk used for cheese is ~25%, but varies widely from ~70–90% in some European countries (Italy, France, Denmark and Germany) to ~0.5% in China. While cheese-like products are produced in most parts of the world, the principal cheese-producing regions are Europe, North America and Oceania. Cheese production has increased consistently over the last two decades at an annual average rate of ~1.5%. As discussed in Chapter 8, this may be attributed to a number of factors including increases in global population and per capita income, globalisation of eating trends/habits, changing lifestyles, growth in use of cheese as an ingredient in the food service (in pizza-type dishes, cheese burgers and salad dishes) and industrial sectors (cordon bleu entrees, co-extruded products with cheese and gratins).

The increase in consumption has been paralleled by a greater emphasis on improved quality and consistency with respect to the levels of particular nutrients (fat, protein, calcium -Ca²+ and sodium -Na+), physical properties (texture and cooking attributes), sensory characteristics and processability (size reduction attributes, such as shredability; ability to yield processed cheeses or other cheese products when subjected to secondary processing). Consequently, this has necessitated an increase in the quality and consistency of all inputs (milk composition/quality, enzyme activity/purity, starter cultures characteristics, for example, acid productivity, phage resistance, autolytic properties and flavour-imparting characteristics) and standardisation of the manufacturing process (cf. Chapter 8). In an overall context, milk quality for cheese manufacture may be defined as its suitability for conversion into cheese and deliver cheese of the desired quality and yield. The current chapter examines milk quality for cheese manufacture and the factors affecting it, together with broad-based strategies for improving quality and consistency.

1.2 Overview of milk composition

Milk consists of protein (caseins and whey proteins), lipid, lactose, minerals (soluble and insoluble), minor components (enzymes, free amino acids, peptides) and water (Table 1.1).

The casein fraction coexists with the insoluble minerals as a calcium phosphate–casein complex. The water and its soluble constituents (lactose, native whey proteins, some minerals, citric acid and minor components) are referred to as serum. During cheese manufacture, the milk is subjected to a partial dehydration, involving controlled expulsion of serum and concentration of fat, caseins (and in some cases denatured, aggregated whey proteins) and some of the minerals. The methods engaged to affect the dehydration include limited destabilisation and aggregation of the calcium phosphate casein in the form of a gel network which

Table 1.1 Compositional and gelation characteristics of cows’ milks.

Source: Compiled from O’Brien et al. (1999b–d), Mehra et al. (1999) and Hickey et al. (2006b) for manufacturing milks.

aBased on the analysis using the Formagraph (Type 1170, Foss Electric, Denmark) on milks at pH 6.55 and rennet-treated at a level corresponding to ~0.18 mL L−1 (Chymax Plus, Pfizer Inc., Milwaukee, WI); RCT is an index of rennet coagulation (gelation) time, A30 of the curd firmness after 30 min, and 1/k20 of gel firming rate.

encloses the fat and serum via specific enzymatic hydrolysis of the casein, acidification (by fermentation of milk lactose to lactic acid by added bacterial cultures), elevated temperature and various mechanical operations as discussed in Chapter 8. Amongst others, the degrees of casein aggregation and dehydration are critical parameters controlling the properties and quality of the final cheese.

Although manufacturing procedures for most cheese types are very defined (at least in large modern cheesemaking facilities) in terms of technology applied and the type and levels of operations imposed on the milk (cf. Chapter 8), variations in cheese quality do occur. Seasonal variation in the composition and quality of milk are considered to be crucial factors contributing to the inconsistency in quality. Consequently, an overview of milk composition in terms of its relevance to cheese manufacture is presented below. The main focus of this chapter is on cows’ milk, which accounts for an estimated 95% of total milk used in cheese manufacture; the characteristics of other milks are discussed elsewhere (Anifantakis, 1986; Juárez, 1986; Remeuf & Lenoir, 1986; Muir et al., 1993a,b; Garcia-Ruiz et al., 2000; Bramanti et al., 2003; Huppertz et al., 2006; Kuchtik et al., 2008; Caravaca et al., 2009).

1.2.1 Casein

The nitrogenous fraction of cows’ milk typically consists of casein, whey protein and non-protein nitrogen (urea, proteose-peptones, peptides) at levels of ~78, 18 and 4 g 100 g−1, respectively, of total nitrogen (Table 1.1).

Casein, which is typically present at a level of 2.5 g 100 g−1 in cows’ milk, is the main structural protein of both rennet- and acid-induced milk gels (Table 1.1). The casein is heterogeneous, comprising four main types: αs1, αs2, β and κ, which represent ~38, 10, 35 and 15 g 100 g−1 of the total casein, respectively (Fox & McSweeney, 1998; Fox, 2003; Swaisgood, 2003). Model studies in dilute dispersions indicate that the individual caseins vary in the content and distribution of phosphate (Table 1.2); the respective number of (serine) phosphate residues per mole of casein are ~8, 10–13, 5 and 1 for αs1-, αs2- β- and κ-caseins, respectively. The serine phosphates bind calcium and calcium phosphate, and consequently, different caseins have different calcium-binding properties. Generally, αs1-, αs2- and β-caseins bind calcium strongly and precipitate at relatively low calcium concentrations (~0.005–0.1 M CaCl2 solutions), inclusive of the calcium level in milk (30 mM); in contrast κ-casein is not sensitive to these calcium concentrations and can, in fact, stabilise up to 10 times its mass of the calcium-sensitive caseins.

Casein in milk exists in the form of spherical-shaped colloid particles (~40–300 nm diameter), known as casein micelles (Fox & Brodkorb, 2008; McMahon & Oommen, 2008). Different models have been proposed for the structure of the casein micelle on the basis of the location of individual caseins (in response to their calcium sensitivity) and the calcium phosphate. These include:

sub-micelle model (Schmidt, 1982), in which sub-micelles are ‘cemented’ together by colloidal calcium phosphate (CCP) and κ-casein-rich sub-micelles are mainly concentrated at the surface of the micelle; the hydrophilic C-terminal region of the κ-casein orient into the serum as a highly hydrated ‘hairy layer’ that is in a state of constant flux and confers stability to the micelle by steric repulsion;

Table 1.2 Characteristics of cows’ milk proteins of relevance to cheese manufacture.

Source: Compiled from Mulvihill & Donovan (1987), Fox & McSweeney (1998), Fox (2003) and Swaisgood (2003).

dual bonding model (Horne, 1998), in which the interior of the micelle is composed of αs- and β-caseins which form a lattice through interactions between hydrophobic regions (hydrophobic-induced) and between hydrophilic regions containing phosphoserine clusters (that attach to CCP clusters), while κ-casein molecules located at the surface interact hydrophobically with the other caseins (αs- or β-) and orient their highly hydrophilic regions (hairs) into the serum;

tangled, cross-linked web model (Holt & Horne, 1996), comprising a ‘tangled’ mass of rheomorphic casein chains cross-linked by calcium phosphate nanoclusters, similar in casein composition throughout but with the chains becoming more diffuse at the micelle periphery (on moving outwards from the dense centre); and

interlocked lattice model (McMahon & Oomen, 2008), featuring a system of interlocking sites composed of anchoring calcium phosphate nanoclusters (several hundred per micelle), which bind the phosphoserine domains of αs- and β-caseins; the hydrophobic ends of these caseins orientate away from the calcium phosphate nanocluster and interact hydrophobically with other α- and β-caseins, while κ-casein is predominantly surface located because of its lack of phosphoserine domains (to bind to the calcium phosphate nanoclusters) and its highly charged C-terminal regions (which prevents strong electrostatic interactions).

In all of the above models, the arrangement of casein within the micelle is such that the interior is mainly occupied by the calcium-sensitive caseins (αs- and β- and κ-casein is principally located at the surface, with its hydrophilic C-terminal region (caseinomacropeptide) oriented outwards toward the serum phase in the form of protruding negatively charged hairs, which create an electrokinetic potential of ~−20 mV and confer stability to the micelle by electrostatic repulsion, Brownian movement and a consequent steric repulsion (de Kruif & Holt, 2003; Horne & Banks, 2004). The κ-casein C-terminal projecting from the micelle surface has been considered as an extended polyelectrolyte brush (de Kruif, 1999), a region containing 14 carboxylic acid groups and immersed in a milk serum with a high ionic strength (~0.08 M) due to the presence of various ions (e.g. potassium, sodium, chloride, phosphate, citrate). Consequently, electrostatic interactions (between the C-terminal regions) at physiological conditions are very short and highly screened (by the high ionic strength). This is conducive to a high degree of ‘solvency’ and extension of the κ-casein C-terminal hairs and to the stability of the micelle as a whole. Moreover, the C-terminal region of the κ-casein is glycoslyated to varying degrees (Table 1.2; Saito & Itoh, 1992; Mollé & Leonil, 1995; Fox & McSweeney, 1998; Mollé et al, 2006), containing galactose, N-acetylgalactosamine (GalNAc) and/or N-actetylneuraminic (sialic) acid (NANA) (Dziuba & Minkiewicz, 1996). These may further enhance the ability of κ-casein to increase micelle stability by steric impedance and electrostatic repulsion via their contribution to increase in water binding (to carbohydrate moieties) and to negatively charged carboxylic groups (on the NANA molecule). O’Connell & Fox (2000) found that the level of glycosylation of κ-casein and protein surface hydrophobicity increased as a function of micelle size.

While a predominant surface location of κ-casein confers stability to the casein micelle in native milk, it renders it susceptible to aggregation/flocculation by processes which reduce the solvency of (and collapse/flatten) the κ-casein hairs or remove them, and thereby enable contact between the more hydrophobic micelle cores, for example cleavage of the κ-casein by acid proteinases, reducing the negative charge by acidification, reducing ionic strength by microfiltration/diafiltration at native pH. However, the interactions between the micelle cores are modified by many factors, including pH, composition of the serum phase, ionic strength, protein concentration and conditions to which milk is subjected (heat, acidification, ultrafiltration/diafiltration homogenisation, shearing).

The casein micelles on a dry weight basis consist of ~7 g 100 g−1 ash (mainly calcium and phosphorous), 92 g 100 g−1 casein and 1 g 100 g−1 minor compounds including magnesium and other salts. They are present in milk at 10¹⁴−10¹⁶ mL−1, are highly hydrated (~3.7 g H2O g−1 protein), are spherical and have a diameter of ~80 nm (100–500 nm), a surface area of ~8 × 10−10 cm² and a density of ~1.063 g cm−3 (Fox & McSweeney, 1998).

1.2.2 Whey protein

Whey protein in cows’ milk is typically ~0.6–0.7 g 100 g−1 and consists of four main types – β-lactoglobulin (β-Lg), α-lactalbumin (α-La), immunoglobulin(s) (Ig) and bovine serum albumin (BSA) at levels of ~54, 21, 14 and 6 g 100 g−1 of total (Table 1.2). The properties of the individual whey proteins have been extensively reviewed (Table 1.2; Mulvihill & Donovan, 1987; Brew, 2003; Fox, 2003; Hurley, 2003; Sawyer, 2003). In milk, they exist as soluble globular proteins and are characterised by a relatively high level of intramolecular disulphide bonding, and β-Lg and BSA each contain one cysteine residue per mole. On heat-induced denaturation, the whey proteins can interact via thiol-disulphide bonds with other whey proteins and with κ-casein. The latter results in the formation of κ-casein/β-Lg aggregates either at the surface of the casein micelle or in the serum phase or both (cf. Chapter 8). The size and location (serum/micelle surface) of these aggregates are affected by severity of heat treatment of milk, pH at heating, ionic strength, calcium level and casein-to-whey protein ratio. The degree of interaction and size/location of aggregates have a profound effect on the structure and physical properties of rennet- and acid-induced milk gels, and hence on cheeses (see Chapter 8). For example, a high level of casein-whey protein interaction, induced by high heat treatment of the milk (e.g. 95°C for ≥ 1−2 min, ~≥40% denaturation of total whey protein; Guinee et al., 1995), is highly favoured in the manufacture of yoghurt and smooth-textured cheeses with a high moisture-to-protein ratio, such as cream cheese and ultrafiltration-produced Quark. In these products it increases protein recovery and moisture binding (reduce syneresis), contributes smoothness and enhances yield (Guinee et al., 1993). In contrast, high heat treatment of milk is unsuitable for acid-curd cheeses with a granular structure (Cottage cheese) or for Quark manufactured using a mechanical separator, as it impedes whey expulsion during separation and makes it difficult to achieve the desired dry matter and texture characteristics. High heat treatment of milk is generally undesirable for rennet-curd cheeses as denatured protein at levels of ≥25% of total (at heat treatments of 82°C for 26 s, or greater) impedes the ability of the milk to gel on rennet addition, causes marked deterioration in melt properties of the cheese (Rynne et al., 2004) and reduces the recovery of fat from milk to cheese (see Chapter 8). However, a higher-than-normal heat treatment that gives a moderate degree of whey protein denaturation may be desirable as a means of modulating the texture of reduced fat cheese, e.g. reduce firmness (Guinee, 2003; Rynne et al., 2004).

1.2.3 Minerals

Cows’ milk contains ~0.75 g 100 g−1 ash, which comprises K+, Ca²+, Cl−, P⁵+, Na+ and Mg²+ at concentrations (mg 100 g−1) of ~140, 120, 105, 95, 58 and 12, respectively (Table 1.2; White&Davies, 1958a; Chapman & Burnett, 1972;Keogh et al., 1982; Grandison et al., 1984; O’Brien et al., 1999c). These minerals are partitioned to varying degrees between the serum (soluble) and the casein (colloidal or insoluble) in native milk (pH ~6.6−6.7) at room temperature. Serum concentrations as a percentage of the total concentration for each of the minerals are ~100, 100, 100, 66, 43 and 34 for Na+, K+, Cl−, Mg²+, P²+ and Ca²+, respectively. The partition concentrations of Ca²+ and P²+ between the colloidal and soluble states in native milk is controlled mainly by the degree of ionisation of the casein (micelle), which in milk may be considered as a very large dominant anion that regulates the degree of binding of the counterion calcium, to an extent affected by the concentration of calcium per se and those of citric acid and phosphate. A major difference between the calcium salts of citrate (tricalcium citrate − Ca3(C6H5O7)2) and phosphate (tricalcium phosphate − Ca3(PO4)2) is their solubility, with the solubility product of the latter being very low (2.07 × 10−33 mol L−1 at 25°C) compared to the former (3.23 × 10−3 mol L−1 at 25°C).

Cows’ milk typically contains ~120 mg 100 mL−1 calcium (~30 mM), which exists as colloidal inorganic calcium (~12.5 mM), caseinate calcium (8.5 mM), soluble unionised calcium (6.5 mM) and serum ionic calcium (2.5 mM). Calcium attached to the casein micelle, referred to as micellar calcium phosphate, is composed of the colloidal inorganic Ca²+ (more frequently denoted CCP) and caseinate Ca²+. The former occurs as a calcium phosphate complex attached indirectly to the organic serine phosphate groups, while the latter is attached directly to casein via the dissociated ε-carboxyl groups of acidic amino acids including aspartic (pKa ~3.9) and glutamic (pKa ~4.1) acids. Owing to the high molarity of glutamic and aspartic acids (~25 and 7 mM) in milk (with a casein content of 2.5 g 100 g−1), it can be inferred that only ~26 g 100 g−1 of the available ε-carboxyl groups are titrated with calcium and that these groups could potentially bind with added calcium to increase the susceptibility of the casein to aggregation, especially on rennet treatment. The sensitivity of the individual caseins to calcium precipitation as found from model studies in dilute solutions varies and tends to increase with the number of moles of both phosphate and glutamic acid per mole of casein. Hence, the concentration of Ca²+ at which the individual caseins precipitate is lowest for αs2-casein (<2 mM), intermediate for αs1-casein (3–8 mM) and β-casein (8–15 mM), and highest for κ-casein, which remains soluble at all of these concentrations and can prevent the precipitation of the other caseins (Aoki et al., 1985).

In the context of the milk salt system, the milk may be viewed as a ‘soup’ consisting of a large colloidal anion (calcium phosphate casein) dispersed in a serum containing various soluble salt and ionic species (calcium citrate, sodium phosphate, potassium and ionic calcium). The insoluble (colloidal salts associated with the casein) and soluble (serum) salts exist in equilibrium. While the soluble citrate and phosphate compete with the casein for calcium ions (resulting in the formation of calcium citrate and insoluble calcium phosphate), the polyvalent casein is the main player controlling the equilibrium concentrations of salts. However, slight changes in pH and concentrations of serum salts (e.g. as a consequence of natural variation or fortification) can affect the equilibrium balance, and consequently the charge and reactivity of the casein.

1.2.4 Milk lipids

Cows’ milk typically contains ~3.7 g 100 g−1 lipid, but the level varies significantly (from ~3.0 to 5.0 g 100 g−1) with breed, diet, health, stage of lactation and animal husbandry. Triacylglycerols, denoted as milk fat, represent ~96–99 g 100 g−1 lipid. The remaining (1–2 g 100 g−1) consists of phospholipids (0.8 g 100 g−1), diacylglycerols, sterols (0.3 g 100 g−1) and trace quantities of carotenoids, fat-soluble vitamins and traces of free fatty acids (FFA) (Jensen, 2002; Huppertz et al., 2009). The fat in milk exists in the form of dispersed globules (~2–6 μm average volume weighted diameter) (Wiking et al., 2004), surrounded by a lipoprotein membrane (milk fat globule membrane, MFGM) (Keenan & Maher, 2006). The MFGM stabilises the enclosed fat against coalescence and fusion (and hence, phase separation) and access from lipases, such as the lipoprotein lipase (LPL) naturally present in native milk, or from lipases of contaminating microorganisms, such as Pseudomonas spp. (Ward et al., 2006). Inadvertent damage of the membrane, as, for example, by manhandling of the milk (e.g. excessive shearing, turbulence, cavitation; see Section 1.5.4), is highly undesirable in cheese manufacture. It leads to free fat in the cheese milk, lower recovery of milk fat to cheese, lipolysis of the fat by lipases that survive pasteurisation treatment, high levels of FFA and undesirable flavours (e.g. bitter, soapiness, metallic), especially in some cheese types (e.g. Emmental, Gouda, Cheddar). In the latter cheeses, only low to moderate levels of FFA are required for satisfactory flavour (Cousin & Marth, 1977; Woo, 1983;Gripon, 1993;Brand et al., 2000; Collins et al., 2004; Ouattara et al., 2004; Deeth & FitzGerald, 2006; see also Chapter 8). Nevertheless, there are a number of applications in cheese manufacture where the cheese milk is homogenised, resulting in physical breakage of the MFGM and its replacement by a newly formed membrane composed of casein and whey proteins, and smaller fat globules (Huppertz & Kelly, 2006). The reformed fat globule, owing to its smaller size (~1.0 μm), is stable to flocculation and creaming, but does not isolate the enclosed fat from lipolytic enzymes. These properties are exploited in the manufacture of cheeses (see Chapter 8):

high-fat acid-curd cheeses, such as Cream cheese, where the smaller fat globules prevent flocculation and creaming during the relatively long incubation/gelation period and where the reformed fat globule membrane enables the fat globule to behave as a fat-filled protein particle, become an integral part of the gel network during acid gelation and contribute to the desired texture characteristics (Guinee & Hickey, 2009; cf. Chapter 8); and

Table 1.3 Free fatty acid profile of milk fat triacylglycerols.

Source: Compiled from Jensen (2002) and MacGibbon & Taylor (2006).

aValues in parentheses indicate the range of values reported in the literature.

rennet-curd cheeses where a high level of lipolysis is desirable (e.g. blue-type cheeses), where added lipases or lipases from secondary starter cultures can access the fat more easily, bring about selective hydrolysis of the triacylglycerols and release the FFA that lead to the desired flavour.

The principal fatty acids in milk fat on a total weight basis are C16:0 (palmitic), C18:1(oleic) and C14:0 (myristic) in decreasing order (Table 1.3). While the shorter chain fatty acids (C4:0 to C12:0) are present in lower quantities on a weight basis, they are primarily responsible for the piquant flavour of hard Italian cheeses, such as Parmesan and Romano, or the sharp goaty/sheep-like flavours of soft goat milk cheeses. These fatty acids are hydrolysed from the milk fat triacylglycerols by lipase enzymes, which gain access owing to damage of the MFGM during cheese manufacture and maturation. The principal sources of these lipases are added exogenous enzymes (added rennet paste, pregastric esterase), secondary flora (Brevibacterium linens, Penicillium roqueforti, Geotrichiun candidum; see also Chapter 6), starter culture lactic acid bacteria and culture adjuncts (Lactococcus spp., Lactobacillus helveticus) (Collins et al., 2004; Hickey et al., 2006b; Santillo et al., 2007; Hashemi et al., 2009; Jooyandeh et al, 2009).

1.3 Principles of cheese manufacture

Cheese is a concentrated protein gel, which occludes fat and moisture. Its manufacture essentially involves gelation of cheese milk, dehydration of the gel to form a curd and treatment of the curd (e.g. dry stirring, cheddaring, texturisation, salting, moulding, pressing). The moulded curd may be consumed fresh (shortly after manufacture, for example within 1 week) or matured for periods of ~2 weeks to 2 years to form a ripened cheese. The gelation of milk may be induced by:

selective hydrolysis of the κ-casein at the phenyalanine105–methionine106 peptide bond by the addition of acid proteinases, referred to generically as rennets (chymosin, pepsin);

acidification (using starter cultures or food-grade acids and/or acidogens), at a temperature of 20–40°C, to a pH value close to the isoelectric pH of casein, i.e. ~4.6; and/or

a combination of acid and heat, for example heating milk at pH ~5.6 to ~90°C.

1.3.1 Rennet-induced gelation

On treatment of milk with chymosin (rennet), the κ-casein is hydrolysed, with the primary cleavage point being the peptide bond phenylalanine105–methionine106, and the liberation of the highly charged, hydrophilic methionine106–valine169 caseinomacropeptide into the milk serum (whey). This results in an effective ‘shaving’ of the hairy layer from the micelle surface, a marked reduction in the negative surface charge to ~−10 mV, and an increase in the attractive forces between, or ‘stickiness’ of, the para-casein micelle surfaces. Consequently, the latter begin to aggregate when sufficient κ-casein is hydrolysed (~80–90 g 100 g−1 of total; Green et al., 1978; Dalgleish, 1979), resulting in the formation of clusters/aggregates of para-casein micelles that fuse gradually and eventually ‘knit’ into a restricted, periodic repeating, solid-like viscoelastic gel network (Fig. 1.1). The enzymatic stage of rennet coagulation and the aggregation of enzymatically altered sensitised para-casein micelles overlap. While the exact contribution of calcium to rennet coagulation is unclear, it is likely that the casein calcium (which in effect may be considered as pre-bound ionic calcium) is the principal agent inducing cross-linking and aggregation of the para-casein micelles into a gel. The serum ionic calcium in milk is in equilibrium with the casein calcium. Hence, apart from reflecting the level of casein-bound calcium, serum ionic calcium probably plays little, or no, direct role in rennet-induced casein aggregation and gelation of milk. Similarly, the progressive increase in gel firmness of rennet-treated milks on the addition of calcium chloride (ionic calcium) while retaining a constant pH (Fig. 1.2) probably reflects the consequent increases in the levels of casein calcium and CCP rather than an increase in the serum ionic ion calcium per se. Hence, it is noteworthy that on concentration of milk by evaporation, the calcium ion activity slightly decreases from ~ 1.0 to 0.75 mM L−1 while the levels of micellar calcium increase (Nieuwenhuijse et al., 1988). Rennet-induced gelation of milk is hindered by a variety of factors, which either:

restrict access of the rennet to its substrate (κ-casein), for example complexation of denatured whey protein with κ-casein at the micelle surface, as a result of high heat treatment of the cheese milk (Fig. 1.1; Guinee, 2003);

act as obstacles to the aggregation and fusion of rennet-treated casein micelles, for example κ-casein/β-Lg appendages at micelle surface, or serum κcasein/β-Lg particles (Guyomarc’h, 2006);

Fig. 1.1 Effect of pasteurisation temperature on changes in storage modulus G’ during the rennet gelation of milk. Note: Milks were heated to various temperatures (in °C) for 26 s prior to rennet addition: 72 (•), 74.6 ( ), 75.9 (°) or 78.5 (Δ); the milks were cooled to 31°C, adjusted to pH 6.55 if necessary with lactic acid solution (5 g 100 g−1), treated with chymosin (Chymax Plus, Pfizer Inc., Milwaukee, WI) at a rate of 0.18 mL of undiluted rennet per litre of milk; all milks had similar contents of protein (3.3 g 100 g−1) and fat (3.4 g 100 g−1); G’ was measured dynamically using low-amplitude strain oscillation rheometry (controlled stress rheometer).

Fig. 1.2 Changes in curd firmness at 60 min (A60; •) and curd firming rate (1/k20; ) of skimmed milk as a function of the level of added calcium chloride. Note: All milk samples (~3.45 g protein 100 g−1 of milk) were adjusted to pH 6.55 prior to measuring the rennet gelation properties at 31°C on the Formagraph (Type 1170, Foss Electric, Denmark); the following parameters were measured k20, a measure of time from the onset of gelation to a output signal width of 20 mm, and A60, the width of the output signal at 60 after rennet addition.

reduce the ‘stickiness’ of rennet-altered casein micelles, for example increased ionic strength (e.g. by the addition of NaCl to the cheese milk as in Domiati cheese) (Awad, 2007; Huppertz, 2007), negative charge (high pH); and/or

reduce the degree of bonding between touching micelles, for example reducing the level of calcium by the addition of ethylenediaminetetraacetic acid (EDTA) or other chelants (Shalabi & Fox, 1982; Mohammad & Fox, 1983; Choi et al., 2007), ion exchange (Mei-Jen-Lin et al., 2006) and/or dialysis (Wahba et al., 1975), or by a naturally low level of Ca²+ as in late lactation milks or milks from cows with subclinical mastitis (White & Davies, 1958a).

Following gel formation, the resultant milk gel is subjected to a number of operations that promote the release of whey, an approximate tenfold concentration of the casein, fat and micellar calcium phosphate components, and a transformation to a curd with much higher dry matter content than the original milk gel (45 g 100 g−1 for Cheddar curd at whey drainage). These operations include cutting the gel into pieces (referred to as curd particles, ~0.5–1.5-cm cubes), stirring and heating the particles in expressed whey, reducing the pH of the aqueous phase inside the curd particle by fermentation of lactose to lactic acid (by the lactic bacteria in the starter culture added to the milk prior to rennet addition), and physical draining of the whey from the curd particles by pumping the curd particle–whey mixture onto perforated screens (cf. Chapter 8). Following whey drainage, the curd particles knit together into a cohesive mass of curd, which is treated to enhance further whey expulsion and concentration to the desired dry matter content of the cheese variety being manufactured; these treatments differ according to variety but typically include further lactose fermentation and pH reduction, cutting the curd mass into pieces (slabs), moulding the pieces to the desired shape and weight of finished cheese, salt addition and pressing. During the dehydration process of the gel, protein concentration and aggregation continues via various types of intra- and intermolecular interactions (Lucey et al., 2003), including calcium bridging (between glutamate/aspartate residues, calcium–CCP bridges between phosphoserine residues), hydrophobic interactions between lipophilic domains and electrostatic interactions (other than calcium bridging). The strength of these interactions is modulated by ionic strength, pH, calcium and temperature, and hydrolysis of proteins to peptides, which alters the hydrophile/lipophile balance of the proteinaceous fraction.

Following manufacture, rennet-curd cheeses are usually matured or ripened by holding under specific conditions of temperature and humidity for periods which range from ~2 to 4 weeks for soft cheeses (for Camembert-type cheeses) to ~2 years for some hard cheeses (for Parmesan-style cheeses). During this period, a host of physico-chemical changes take place which transform the ‘rubbery/chewy’-textured fresh cheese curd to the finished cheese with the desired variety quality characteristics, for example a soft, smooth, short and adhesive texture with a mushroom-like flavour and creamy mouth-feel for Camembert, or a long, elastic sliceable texture and mild, sweet flavour for Leerdammer cheese. These physico-chemical changes include:

glycolysis, conversion of residual lactose to lactic acid by the starter culture and of lactic acid to other compounds, such as acetic acid and propionic acid by secondary starter cultures such as Propionobacteria freudenreichii subsp. shermanii in Emmental-style cheese;

proteolysis, hydrolysis of caseins to peptides and free amino acids by proteinases and peptidases present in the cheese (residual rennet; plasmin, and proteinases and peptidases from the cells of starter culture and non-starter lactic acid bacteria); and

lipolysis, involving the hydrolysis of triacylglycerols to FFA, di- and monoacylglycerols by lipases and esterases from various sources, including native milk LPL, added pregastric esterases and/or secondary cultures.

The physico-chemical and biochemical changes that occur during ripening are discussed in Chapter 8 and several comprehensive reviews are available (Collins et al., 2004; McSweeney & Fox, 2004; Upadhyay et al., 2004; Kilcawley, 2009).

Of particular interest in relation to milk composition and cheese quality is the impact of the proportion of intact αs1-casein content in milk on casein aggregation, strength of the rennet- induced milk gel and texture of the final cheese. The sequence of residues 14–24 is a strongly hydrophobic domain and confers intact αs1-casein with strong self-association and aggregation tendencies in the cheese environment (Creamer et al., 1982); interestingly, this domain also has 3 mol of glutamate, which are expected to contribute to intra- and intermolecular calcium bridges. It has been suggested that self-association of αs1-casein in cheese, via these hydrophobic ‘patches’, leads to extensive cross-linking of para-casein molecules and thus contributes to the overall continuity and integrity of the casein matrix in the cheese curd (de Jong, 1976,1978; Creamer et al., 1982;Lawrence et al., 1987). The early hydrolysis of αs1-casein at the phenylalanine23-phenylalanine24 peptide bond, by residual rennet retained in the cheese curd following manufacture (~10% of that added), results in a marked weakening of the para-casein matrix and reductions in fracture stress and firmness of the cheese during maturation (deJong, 1976,1977; Creamer & Olson, 1982; Malin et al., 1993;Tunick et al., 1996; Fenelon & Guinee, 2000). This hydrolysis is a key step in mediating the conversion from a fresh rubbery curd to a mature cheese with the desired textural and cooking properties (meltability) (cf. Chapters 7–10).

1.3.2 Acid-induced gelation

The caseins in milk are insoluble at their isoelectric points (pH ~4.6) at temperatures >~8°C (Mulvihill, 1992). This property is exploited in the formation of acid-curd cheeses, such as Cottage cheese, Quark and Cream cheese, the manufacture of which involves slow quiescent acidification of the cheese milk to pH ~4.6–4.8 by starter culture, acidogens (e.g. glucono-δ-lactone) at temperatures of 20–30°C (Guinee et al., 1993; Lucey & Singh, 1997; Fox et al., 2000; Farkye, 2004; Lucey, 2004; Schulz-Collins & Zenge, 2004). Acidification results in a number of physico-chemical changes promoting hydration/dispersion or dehydration/aggregation effects on the casein micelle, with the ratio of these effects changing as the pH declines during the acidification (fermentation) process. Reducing the pH from 6.6 to ~5.2–5.4 results in a decrease in the negative charge of the micelles due to titration of negative charges with H+ ions. Nevertheless, this is generally not accompanied by the onset of gelation because of:

solubilisation of micelle ‘cementing’ agent CCP (fully soluble at pH ~5.2 at 20°C);

diffusion of all caseins from the micelle to the serum (owing to a decrease in the degree of electrostatic interaction between phosphoserine residues of αs- and β-caseins and the CCP nanoclusters);

increases in ionic strength of the serum phase; and

hydration of the casein micelles.

However, further reduction in pH in the range ~5.2–4.6 results in aggregation of casein and gel formation, as forces promoting dispersion of casein micelles are overtaken by the sharp reductions in the negative charge and hydration of the casein, the collapse in steric effect associated with the κ-casein C-terminal ‘hairs’ and the increase in hydrophobic interactions. The onset of gelation typically occurs at pH ~5.1 and further reduction in pH toward 4.6 coincides with the eventual formation of a continuous gel structure with sufficient rigidity to enable separation of whey from the curd by physical means (e.g. breakage, stirring, and whey drainage, or centrifugation). The increase in gel rigidity coincides with a sigmoidal increase in the elastic shear modulus of the gelling cheese milk, as the pH continues to decrease towards 4.6 during incubation and the fermentation of lactose to lactic acid (Fig. 1.3).

High heat treatment of the cheese milk (e.g. 95°C for ≤1 min) leads to an increase in the pH at the onset of gelation (from ~4.7 to 5.3) and the rigidity of the resultant gel (cf. Chapter 8; Vasbinder et al., 2003; Anema et al., 2004). These changes coincide with increases in the level of whey protein denaturation and its covalent interaction with κ-casein,

Fig. 1.3 Effect of milk treatment (heated to 90°C for 30 min − •, or unheated − °) on changes in modulus G’ during acid gelation of milk. Note: The milks (1.5 g fat 100 g−1, 3.2 g protein 100 g−1)were cooled to 40°C and a thermophilic starter culture (Streptococcus thermophilus) was added at a rate of 2.5 g 100 g−1; G’ was measured dynamically using low-amplitude strain oscillation rheometry (controlled stress rheometer).

via thiol-disulphide interchange. This interaction occurs both at the surface of the micelle, resulting in the formation of filamentous appendages projecting from the micelle surface, as well as in the serum phase when κ-casein dissociates from the micelle into the serum and interacts with β-Lg to form soluble complexes that sediment as the pH is reduced during fermentation and/or rennet treatment. The different types of interactions are influenced by pH and the level of whey proteins (Donato & Guyomarc’h, 2009; cf. Chapter 8). In situ denatured whey protein increases the concentration of gel-forming protein, the spatial uniformity of the gel matrix and the number of stress-bearing strands in the matrix. Denatured whey proteins, whether in the form of filamentous appendages (κ-casein/β-Lg) that occur at the surface of micelle and ‘flatten’ on pH reduction or that occur as serum-soluble κ-casein/β-Lg particles that sediment on pH reduction (and/or rennet treatment), act as obstructions that physically obstruct/prevent a high level of interaction of the native casein micelles and, thereby, lead a more continuous gel structure with higher rigidity. The increase in micelle size resulting from complexation with denatured whey protein (Anema & Li, 2003) is conducive to an earlier touching of the casein micelles during the acidification/gelation process and the onset of gelation at a higher pH. The changes in gel structure associated with high heat treatment of milk lead to significant increases in the stiffness (G’) and visual smoothness of the resultant acid gel (Fig. 1.3), a principle which has long been exploited in the manufacture of yoghurt.

In the manufacture of acid-curd cheeses, the milk gel is cut or broken, and whey removal is achieved by various means including centrifugation, ultrafiltration and/or straining the broken gel in muslin cheese bags. In some varieties (e.g. cream cheese), whey separation is further enhanced by heating the broken gel to temperatures of ~80°C prior to centrifugation or to ~50°C prior to ultrafiltration or straining. Treatments of the curd differ with cheese variety. In the manufacture of Quark, the temperature of the concentrated curd (~18 g 100 g−1 dry matter) is cooled rapidly to <8°C by passing through suitable heat exchangers so as to limit hydrophobic interactions between the proteins and to, thereby, minimise the likelihood of defects associated with excessive protein interaction in the final cheese, for example sandy, chalky, or grainy mouth-feel, and/or wheying-off. In contrast, the manufacture of Cream cheese involves high heat treatment of the curd (~80°C), the addition of NaCl (~0.5 g 100 g−1) and hydrocolloids (blends of xanthan/guar gums: ~0.3 g 100 g−1), mixing, homogenisation and cooling. The added hydrocolloids, hydrated and dispersed at high temperature, increase the viscosity of the hot molten cheese curd and reduce the growth of protein aggregates and the occurrence of chalky/grainy texture.

The degree to which the attributes (stiffness, structure) of the gel prior to whey separation and concentration influence the characteristics of the final acid-curd cheese is influenced by the type and extent of operations (whey separation method, heat treatment, type/level of hydrocolloid) following gel formation; this subject is beyond the scope of this chapter and the reader is referred to earlier reviews (Guinee et al., 1993; Lucey et al., 2003; Farkye, 2004; Schulz-Collins & Zenge, 2004; Guinee & Hickey, 2009).

1.4 Quality definition of milk

In an overall context, the quality of milk for cheese may be defined as its characteristics that fulfil the requirements of its users – direct (the cheese manufacturer) and indirect (the cheese user, consumer) (Peri, 2006). The quality requirements may be defined as:

safety, which denotes the absence of associated risk (e.g. pathogenic microorganisms, ‘toxic’ residues) in milk from consuming the cheese from which it is made;

compositional/nutritional, which indicate the conformity to minimum levels of particular components (fat, protein, casein, calcium) that make it suitable for cheese manufacture, for example enable the milk to form a gel suitable for cutting within a certain time after addition of rennet; to give desired manufacturing efficiency (percentage recovery of fat and casein; product yield), composition (levels of protein, calcium, moisture) and sensory characteristics;

microbiological, ensuring that total bacterial count does not exceed a maximum value so as to reduce the risk of the milk quality (level of intact casein, absence of rancidity associated with hydrolysis of milk fat) being compromised in terms of its cheesemaking capacity (rennet coagulability, altered levels of pH at different stages of manufacture), cheese yield efficiency (recoveries of fat and casein, cheese yield) and cheese quality (flavour and physical properties);

sensory and functional, implying its possession of the desired hedonic (absence of taints) and physico-chemical characteristics (coagulability by rennet under defined conditions), enabling it to be satisfactorily made into cheese with the desired hedonic (taste, smell), usage (techno-functional) and nutritional characteristics; and

ethical, in terms of its naturalness (non-adulterated) and its compliance to production standards including those pertaining to animal breeding, animal welfare and agricultural/husbandry systems.

Requirements of the first four aspects either can be quantified directly by tests (microbiological, chemical, physical) undertaken by the cheese manufacturer or regulatory agencies, or can generally be perceived by both the manufactures and users of the milk, as they may impact on cheesemaking capacity of the milk, yield efficiency or product quality. Generally, ethical requirements (apart from adulteration) cannot be tested and/or perceived directly by the users; for example, analysis of milk or consumption of the resultant cheese cannot verify that the milk was produced in compliance with organic farming methods. Compliance to ethical requirements is generally considered to be fulfilled by the milk producer and, moreover, is ensured by specifications set by government agencies (EU, 1992, 2004) and organisations such as dairy cooperatives and organic milk supplier organisations.

In the current chapter, milk quality for cheese manufacture will be discussed under the following criteria, each of which involves different types of sub-criteria or characteristics.

1.4.1 Safety/public health (pathogens including Mycobacterium tuberculosis, Brucella spp., toxic residues, and contaminants)

Directive 92/46 (EU, 1992) specifies that raw milk must come from healthy animals and should not endanger human health by way of infectious diseases or foreign substances that are communicable to human beings through the milk. A recent study has attributed 9% of foodborne disease cases to milk consumption (Adak et al., 2005).

Pathogenic bacteria

The presence of potentially pathogenic bacteria in milk is well documented (Rea et al., 1992; Jayarao & Henning, 2001). The pathogens reported as the most common agents implicated in milkborne disease include Salmonella spp.,Campylobacter spp. and Escherichia coli (Gillespie et al., 2003), but others found in milk could also have public health implications, such as Mycobacterium tuberculosis and Listeria monocytogenes (Jayarao & Henning, 2001). Reed and Grivetti (2000) reported that surveys on Californian dairies revealed the presence of a variety of bacteria that could make people ill, and raw milk consumption has often been associated with foodborne epidemics due to pathogens, such as Campylobacter spp., Listeria spp. and Salmonella dublin. These microorganisms may enter the mammary gland and thus the milk, from the external environment through the teat orifice during the milking process or during the interval between milkings. Contamination of the external surface of the teat with faecal and other environmental organisms is scarcely avoidable, but is minimised by compliance to the highest standards of hygiene at milking. However, if initial contamination levels are low and subsequent milk storage conditions (hygiene and temperature) are correct, then further bacterial growth will be minimised.

Mycobacterium bovis

This organism has a broad host range and is the principal agent of tuberculosis in wild and domestic animals. This organism can also infect humans causing zoonotic tuberculosis. The transmission of tuberculosis to humans in the United Kingdom following consumption of unpasteurised milk was reported by de la Rua-Domenech (2006). Brucella spp. are pathogens, which are highly infectious and capable of causing disease in both animals and humans. The pathogenic strain Brucella abortus is more associated with cows, whereas Brucella melitensis is more commonly found in sheep and goats. Transmission to humans can be (amongst other routes) via milk and milk products (Gupta et al., 2006).

Regulation 853/2004 (EU, 2004) (Annex III, Section IX) states that raw milk must come from animals that do not show symptoms of infectious diseases communicable to humans through milk. In particular, as regards tuberculosis and brucellosis, this regulation states that raw milk must come from cows (or buffalos) belonging to a herd which, within the meaning of Directive 64/432 (EU, 1964), is free or officially free of tuberculosis and brucellosis, and if not, the milk may only be used with the authorisation of the competent authority. In addition to compliance with directivities on milk quality, perhaps the most effective means of ensuring the safety of milk from a public health perspective may be to implement ongoing training of dairy farmers and their employees in the areas of cow management, milk handling and storage procedures, fundamentals of toxin and disease transmission, and pathogen effects on human health. In addition, pasteurisation of milk represents possibly the most significant and successful contribution to milk safety (Holsinger et al., 1997).

Toxic residues/contaminants

These compounds in the animal’s body may be shed into milk and thus pose a threat to human health. Chemical residues are remnants of purposeful additions to the food chain (see Section 1.5.5), whereas contaminants represent any biological or chemical agent and any other foreign substances (e.g. dioxins, pesticides) that could gain entry to the milk and, as a result, compromise food safety or suitability for use. The most common chemical residues found in milk are antibiotics, following administration to treat mastitis. Regulation 853/2004 (EU, 2004) (Annex III, Section IX) states that raw milk must come from animals to which no unauthorised substances have been administered and that where authorised products or substances have been administered, the withdrawal periods for those products have been observed. The most effective means of controlling toxic residues/contaminants is by legislation, voluntary codes of practice, monitoring and surveillance of animal feeds, and prudent use of all animal inputs (Buncic, 2006).

1.4.2 Composition (protein, casein, fat, total solids, lactose, and mineral)

Regulation 2597/97 (EU, 1997) outlines marketing standards to guarantee compositional quality of non-standardised whole milk, and include minimum fat and protein concentrations (g 100 g−1) of 3.5 and 2.9 (based on a fat content of 3.5 g 100 g−1), respectively. The specific combination of milk characteristics required for cheese depends on the type of cheese being manufactured. For example, sheep’s milk is more suited than cows’ milk for the production of piquant-flavoured cheeses, such as Pecorino Romano owing to the higher concentration of short-chain fatty acids (C4:0, C6:0, C8:0, C10:0 and C12:0) in its milk fat, which contributes to this flavour profile (Nelson et al., 1977; Lindsay, 1983; Woo & Linday, 1984; Medina & Nũnez, 2004). The low carotenoid content of sheep’s and goat’s milk relative to cows’ milk is also more suited to the manufacture of white-coloured cheese varieties, such as Manchego and Roquefort cheeses (Anifantakis, 1986; Fox et al., 2000). However, cows’ milk can vary dramatically in carotenoid content from ~4 to 13 μg g−1 fat, depending on breed, feed type and stage of lactation (Noziere et al., 2006; Calderon et al., 2007). In contrast, sheep’s milk because of the above characteristics is unlikely to be suitable for the manufacture of Cheddar cheese, in which the rich straw-yellow colour, relatively low level of lipolysis (Hickey et al., 2006a,b, 2007) and non-rancid flavour are key quality criteria. Owing to its low ratio of αs1- to αs2-casein, goat milk gels much more slowly than cow milk on rennet addition and forms markedly weaker gels and curds, and is consequently much more suited to the manufacture of soft cheese (Storry et al., 1983; Juarez & Ramos, 1986; Medina & Nunez, 2004), but much less so to the large-scale manufacture of hard cheeses, such as Emmental, Gouda, Mozzarella and Cheddar. Apart from the altered proportions of individual caseins, other factors such as the (generally) lower contents of calcium and total casein may also contribute to the relatively poor rennet coagulation characteristics of goat milk.

Optimising manufacturing procedures for milks of varying compositions

Rennet-curd cheese is a product created through controlled enzymatic destabilisation and aggregation of colloidal calcium phosphate casein micelles in the form of a calcium phosphate para-casein gel, enclosing fat and moisture. The gel is subjected to various operations (e.g. breaking/cutting, pH reduction, temperature elevation) to induce expulsion of whey and transition from a low-solids gel to a high-solids cheese curd. During this dehydration, involving breakage and shrinking of the gel, the gel/matrix structure continually rearranges, resulting in further aggregation and fusion of the para-casein. The compositional characteristics of good quality milk for the manufacture of all cheeses are those that enhance this controlled aggregation under optimised cheesemaking conditions to give an acceptable manufacturing time, cheese with the desired composition, high yield and excellent quality.

However, a given set of milk compositional characteristics may not fulfil all three requirements simultaneously unless the manufacturing procedure is optimised. For example, the potential of milk with a higher than normal intact casein content to deliver a high yield of cheese with the desired composition and quality may not be realised if the standard operating procedure (SOP) was developed using milks with lower casein content. A critical step in the SOP for any cheese recipe is the firmness of the gel at cut, which can affect the cheese moisture, pH, salt in moisture, yield and quality (cf. Chapter 8). Yet in most modern cheesemaking operations, rennet is added to the milk on a volume basis (rather than on basis of casein load: volume × concentration) and the gel is cut at a fixed time after rennet addition (rather than on the basis of firmness). While such a process may appear to be standardised (fixed rennet dosage per volume of milk, fixed set-to-cut time), it automatically promotes variable curd firmness at cutting when the properties of the cheese milk (e.g. casein number, casein content, pH, calcium content) presented to the SOP change seasonally. Such SOPs are frequently established by the investigations of production support personnel, working over relatively short-time periods on milks with composition parameters falling within a narrow range. However, seasonal variations in milk composition can be relatively large; for example, in Ireland, protein can vary from ~3.1 to 3.8 g 100 g−1 in milk from pasture-fed, spring-calved herds (Mehra et al., 1999; O’Brien et al., 1999d; Guinee et al., 2006). Significant seasonal changes in milk composition are also common elsewhere, including the United Kingdom (Grandison 1986; Banks & Tamime, 1987), France (Martin & Coulon, 1995), New Zealand (Auldist et al., 1998; Nicholas et al., 2002), Australia (Auldist et al., 1996; Broome et al., 1998a; Walker et al., 2004) and Canada (Kroeker et al., 1985). Hence, there is a need to standardise basic parameters, such as protein-to-fat ratio, casein content (ideally), ratios of starter culture and rennet to casein load, starter culture activity, firmness at cut and the pH at different stages of manufacture (e.g. at set) to achieve the optimum performance from good quality milk. Using such an approach to develop SOPs should minimise seasonal variations in cheese composition, manufacturing efficiency, biochemical changes during maturation and quality (cf. Chapter 8). The use of the most-up-to-date technology (including milk casein standardisation), process modelling, in combination with on-line monitoring (in-vat curd firmness sensors), is seen as an approach for further optimisation of process control and improvement in cheese quality.

Effects of variations in different compositional parameters

The effects of many compositional parameters of milk on cheese manufacture (rennet coagulation characteristics), cheese yield and/or cheese quality have been investigated (Okigbo et al., 1985a-c; Guinee et al., 1994, 1997, 2006; Broome et al., 1998a,b; Auldist et al., 2004; Mei-Jen-Lin et al., 2006; Wedholm et al., 2006; Jõudu et al., 2008) and are summarised in Table 1.4. Generally, numerically higher values of the following variables are positively correlated with enhanced rennet coagulation properties (more rapid curd firming rate, higher curd firmness and shorter set-to-cut time in manufacture) and cheese yield: casein number; contents of total casein, individual (αs1-, β- and κ-) caseins, β-Lg, calcium;

Table 1.4 Characteristics of milk ex-farm important for cheese manufacture.

aND, not defined.

bAminomethyl cumarin.

and ratios of κ-casein to total casein and to individual (αs2- and β-) caseins. For a given rennet-to-casein ratio, the positive effect of the increases in the above milk characteristics on rennet coagulation and/or cheese yield are consistent with a higher concentration of gel-forming casein and/or enhanced aggregation via calcium bridges, calcium phosphate bridges and hydrophobic interactions. The positive effect of a high κ-casein-to-total casein ratio is expected because of:

the presence of three hydrophobic domains and a high level of aspartic acid (4 mol) on the N-terminal (AA1−20) region of the para-κ-casein;

the reduction in casein micelle size that generally accompanies an increase in the ratio of κ-casein to total casein (Dalgleish et al., 1989; Umeda & Aoki, 2002);

the relatively high hydrophobicity of the para-κ-casein, which would enhance the aggregation of the rennet-altered micelles.

However, it is noteworthy that while an increase in the proportion of κ-casein to total casein has been found to enhance the rennet gelation characteristics of milk, it has been found to give a non-significant decrease in the yield of laboratory-scale cheeses (Wedholm et al., 2006); this contrasts with the results of studies on the influence of genetic variants of κ-casein and β-Lg, which show that the B-alleles of these proteins have, in addition to other factors (casein micelle size), higher levels of κ-casein as a percentage of total casein. While a higher κ-casein, as a percentage of total casein, may coincide with a higher loss of caseinomacropeptide, care must be taken when interpreting results on cheese yield as affected by any parameter, owing to the confounding effects of indirect variables (e.g. variation in firmness of gel at cut, moisture content of curd).

The genetic variant of κ-casein has a major influence on cheesemaking properties of milk, with κ-casein BB variant giving superior rennet coagulation characteristics, fat recovery from milk to cheese and cheese yield capacity compared to milk having the κ-casein AB, which in turn is superior to milk with the corresponding AA or AE genotypes (van den Berg et al., 1992; Walsh et al., 1995,1998a; Ng-Kwai-Hang & Grosclaude, 2003; Wedholm et al., 2006). Reported increases in moisture-adjusted cheese yield with the κ-casein BB variant, compared to κ-casein AA variant range from ~3 to 8%, depending on milk composition and cheese type. Generally, the κ-casein AB variant has been found to exhibit rennet coagulation and cheese-yielding characteristics that are intermediate between those of κ-casein AA and BB. The superior rennet coagulation and cheese-yielding characteristics of the κ-casein BB variant compared to the AA variant appear to be related to its higher casein content, higher level of κ-casein as a percentage of total casein, smaller micelles and lower negative charge. These properties are conducive to a higher degree of casein aggregation and a more compact arrangement of the para-casein micelles, which in turn favours more numerous intermicellar bonds during gel formation. Indeed, it has been shown using model rennet coagulation studies that for a given casein concentration, the curd firming rate of rennet-treated micelle suspensions was inversely proportional to the cube of the micelle diameter (Horne et al., 1996).

In milk there is an inverse relationship between the concentrations of lactose and chloride, which is the basis of the test for Koestler number, to distinguish between normal and abnormal (e.g. mastitic) milks (Ferreiro et al., 1980; Horvath et al., 1980; Fox & McSweeney, 1998)

where a value of <2 is normal and >2.8–3.0 is abnormal. Mastitis increases the concentrations of Na+, K+, Cl− ions but decreases the concentration of lactose in the milk, as a response to maintain osmotic pressure within the mammy gland system. While a high level of Cl− (or Na+, K+) per se probably has little direct negative impact on para-casein aggregation and curd formation, apart from giving a slight increase in ionic strength, its occurrence is indicative of high somatic cell count (SCC) (250 to >400 × 10³ and >1000 × 10³ cells mL−1for subclinical and clinical mastitis, respectively). Elevated SCC results in a marked increase in γ-caseins, proteose-peptones and the ratio of soluble to micellar casein (Anderson & Andrews, 1977; Ali et al., 1980a,b; Schaar 1985a; Saeman et al., 1988). These changes ensue from hydrolysis of β- and αs2-caseins by the elevated activity of plasmin (and probably other proteinases) in the milk; κ-casein is hydrolysed more slowly by plasmin than β- and αs2-caseins. The ensuing decrease in the intact casein level reduces the degree of casein aggregation as reflected by a marked deterioration in rennet gelation properties, syneretic properties and cheese yield (Donnelly et al., 1984; Okigbo,et al., 1985a; Mitchell et al., 1986; Politis & Ng-Kwai-Hang, 1988a–c; Barbano et al., 1991; Barbano, 1994; Auldist et al., 1996; Klei et al., 1998). An increase in SCC from 1 × 10⁵ to >5 × 10⁵ cells mL−1 typically results in a reduction of ~3–7% in the moisture-adjusted (to 37 g 100 g−1) yield of Cheddar cheese. However, it is noteworthy that the decrease is also relatively large (~0.4 kg Cheddar cheese 100 kg−1 milk) on increasing the SCC from 1 × 10⁵ to 2 × 10⁵ cell mL−1, a range that would be considered relatively low for good quality bulk milk. Losses of fat and protein during Cheddar cheese manufacture increased, more or less linearly, by ~0.7 and 2.5 g 100 g−1, respectively, with SCC in the range 1 × 10⁵ to 1 × 10⁶ cells mL−1 (Politis & Ng-Kwai-Hang, 1988a,c).

1.4.3 Microbiology (total bacterial count)

Microbial contamination of milk can occur pre-milking as a consequence of animal infection or during, or post-milking as a consequence of direct contact with bacteria in the environment or milk handling equipment and/or, for example, milking machine, on-farm storage, transport. Directive 92/46 (EU, 1992), which became effective from 1 January 1994, contained animal health requirements for raw milk, hygiene requirements for registered holdings and hygiene requirements for milking, collection and transport of milk to collection centres. A package of new hygiene regulations was adopted in April 2004 by the European Parliament and the Council (Regulation 853/2004) (EU, 2004). These became applicable from January 2006, and in the case of milk and milk products, these replace Directive 92/46 (EU, 1992). The new regulations are binding in EU Member States without the necessity of national legislation to be enacted to implement their provisions. However, instead of all of the hygiene requirements being incorporated in a single piece of legislation, the requirements for the dairy sector are contained in three main regulations. One specific Regulation 853/2004 (EU, 2004) lays down specific hygiene rules for food of animal origin, with Annex III (Section IX) containing specific requirements for raw milk and dairy products. Specifically, with regard to plate count standards, milk-processing operators must ensure that raw milk meets the following criteria:

plate count at 30°C ≤100 × 10³ colony forming units (cfu) mL−1 for cows’ milk, corresponding to the rolling geometric average over a 2-month period, with at least two samples per month;

plate count at 30°C ≤150 × 10⁴ cfu mL−1 for milk from other species, corresponding to the rolling geometric average over a 2-month period, with at least two samples per month;

plate count at 30°C ≤50 × 10⁴ cfu mL−1 for raw milk from species other than cows when to be used for manufacture of products using processes that do not involve pasteurisation, corresponding to the rolling geometric average over a 2-month period, with at least two samples per month.

1.4.4 Sensory (appearance, colour, smell, and taste)

Sensory analysis may be used to test the characteristics of milk and may be considered as part of the overall quality control of the product. The sensory attributes of appearance and aroma are important factors in determining the quality of milk. Factors that influence the sensory evaluation of cows’ milk include cow health and feed and the absorption of foreign flavours after milking (Ishler & Roberts, 1991). Flavour defects that are chemically induced (rancidity – specific chemical flavour) cannot be removed or improved, and may become more pronounced on storage (Mounchili et al., 2005). For example, off-flavours in milk may arise as a consequence of improper milking practices (inadequate removal of teat disinfectant prior to milking) and milk handling procedures (excessive agitation leading to free fat) and reduce consumer acceptability. Hence, milk with good sensory characteristics may be maintained by: (a) control of cow diet, (b)