Hard cheeses are ripened after manufacture for periods ranging from a few months to 2 or more years and it is during this ripening period that the flavour and texture characteristic of the variety develop [54, 88]. Cheese ripening usually involves changes to the microflora of the cheese [54, 56], often death and lysis of starter cells, the development of an adventitious non-starter microflora  and, in certain cases, the growth of secondary organisms [117, 128, 137, 142]. It is often difficult to differentiate between the flavours of freshly made curds of different types of hard cheeses immediately after manufacture. It is during ripening that flavour develops as a consequence of microbial and enzymatic changes to residual lactose and to lactate and citrate, liberation of fatty acids (lipolysis) and their subsequent metabolism to volatile flavour compounds and hydrolysis of the casein matrix of the cheese to a wide range of peptides and free amino acids (proteolysis) followed by catabolism of amino acids to further important volatile flavour compounds. Cheese texture is influenced greatly by manufacture which largely determines the moisture content of the cheese  and its calcium and fat and fat-in-dry-matter levels. However, texture changes during ripening due to solubilisation of calcium phosphate , hydrolysis of the casein matrix, changes to water binding within the curd and loss of moisture caused by evaporation from the cheese surface.
While certain changes during the ripening of hard and semi-hard cheeses are always considered defects (e.g. late gas blowing ), others are considered problems only if they exceed certain limits. For example, very low levels of bitterness are normal in the flavour profile of cheeses such as Cheddar and are not considered a defect unless levels of bitter peptides exceed certain limits . Likewise, lipolysis occurs during the ripening of all hard and semi-hard cheeses but the levels of lipolysis characteristic of an Italian Pecorino variety would be considered a defect in Cheddar . Hence, balanced ripening is essential to the quality of hard and semi-hard cheeses.
88 How does flavour develop in cheese during ripening?
It is now generally accepted that there is no one cheese flavour compound and that the flavour of a particular variety stems from the combination of a wide range of volatile and non-volatile compounds present in the correct balance and concentration (the 'component balance theory' of cheese flavour). The biochemistry of cheese ripening is extremely complex and has been a very active area of research in recent years. The reader's attention is drawn to the many reviews of aspects of cheese ripening, and space here permits only the briefest overview of the subject. Conventionally, the biochemistry of cheese ripening is often discussed under three broad headings: (i) metabolism of residual lactose and of lactate and citrate, (ii) lipolysis and metabolism of fatty acids and (iii) proteolysis and amino acid catabolism (Fig. 1).
Most lactose in milk is lost in the whey during cheese manufacture  and the low levels of lactose trapped in the curd are metabolised quickly by starter activity before salt-in-moisture reaches an inhibitory level  or by non-starter lactic acid bacteria (NSLAB) . Lactate produced by starter activity is an important starting point for a range of pathways that contribute positively or negatively to cheese flavour. L-Lactate may be racemised to DL-lactate by NSLAB activity, which may be of significance to the development of Ca-lactate crystals in cheese . Lactate is also the starting point for an anaerobic fermentation by Clostridium spp. leading to late gas blowing . However, lactate metabolism is of great importance to Swiss cheese where it is metabolised by Propionibacterium freudenreichii during the hot-room step of ripening to propionate, acetate, CO2 and H2O . This secondary fermentation is of great significance to the flavour of Swiss-type cheeses and is essential for eye development. In surface mould-ripened varieties such as Camembert and Brie, lactate metabolism by Penicillium camemberti deacidifies the cheese surface with a major impact on cheese texture [128, 132, 133].
Milk fat contains high levels of short-chain fatty acids which, when liberated by lipolysis , are highly flavoured. Levels of lipolysis vary widely between varieties and levels expected and desirable in one cheese may be considered a serious defect in another variety . Cheeses with the highest levels of lipolysis are those that contain an active source of lipases such as Blue cheese  (enzymes from Penicillium roqueforti) or cheeses (e.g. Italian Pecorino varieties, Provolone or traditional Greek Feta) the milk for which is coagulated using rennet paste which contains pre-gastric esterase . Cheeses made from raw milk generally develop higher levels of lipolysis than cheeses of the same variety made from pasteurised milk since the indigenous lipoprotein lipase in milk is largely inactivated by pasteurisation . Starter or non-starter lactic acid bacteria are weakly lipolytic but they are present at high numbers for long periods of ripening and their enzymes contribute to the low levels of lipolysis characteristic of varieties such as Cheddar or Gouda. Likewise, P. freudenreichii contributes, together with the thermophilic starter, to the low level of lipolysis in Swiss cheese.
P. camemberti and the complex Gram-positive bacterial surface microflora of smear-ripened cheese  also produce lipases that contribute to lipolysis in certain varieties. Fatty acids, particularly short chain acids, have a direct impact on cheese flavour but they also act as starting points for another series of reactions leading to the production of thioesters, ethyl esters, 7- or ¿-lactones and, of particular importance to Blue cheese flavour, alkan-2-ones (methyl ketones).
Proteolysis is the most complex and perhaps the most important of the three primary biochemical events that occur during ripening. In most varieties, the caseins are initially hydrolysed by enzymes from the coagulant [27, 28], and to a lesser extent from the milk (plasmin and perhaps somatic cell proteinases ) forming large and intermediate-sized peptides. The latter peptides are degraded further by the cell envelope-associated proteinase and the wide range of peptidases of lactic acid bacteria, ultimately to free amino acids . Proteolysis in hard cheese thus leads to the production of a wide range of peptides (perhaps over 300 in Cheddar) of different sizes and a pool of free amino acids. Peptides may have a direct impact on cheese flavour (some are bitter ) or may provide a brothy background flavour to cheese. Recent research has suggested that the major role of proteolysis in the development of cheese flavour is the production of free amino acids which are the starting points for a series of pathways ('amino acid catabolism') leading to the production of many important volatile flavour compounds in cheese. Catabolism of most amino acids appears to be initiated by the action of an aminotransferase which transfers the amino group to an acceptor molecule, usually a-ketoglutarate, thus forming glutamic acid and a new a-keto acid corresponding to the amino acid being degraded. The a-keto acids are then degraded by a number of pathways to various flavour compounds. Methionine is the principal sulphur-containing amino acid in cheese and its side chain is the source of many important volatile flavour compounds.
COLLINS, Y.F., McSWEENEY, P.L.H. and WILKINSON, M.G. (2004). Lipolysis and catabolism of fatty acids in cheese, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 374-389. CURTIN, A.C. and McSWEENEY, P.L.H. (2004). Catabolism of amino acids in cheese during ripening, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 436-454. McSWEENEY, P.L.H. (2004a). Biochemistry of cheese ripening. Int. J. Dairy Technol. 57, 127-144.
McSWEENEY, P.L.H. (2004b). Biochemistry of cheese ripening: introduction and overview, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 347-360. UPADHYAY, V.K., McSWEENEY, P.L.H., MAGBOUL, A.A.A. and FOX, P.F. (2004). Proteolysis in cheese during ripening, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 392-433.
89 How can the problem of bitterness in cheese be solved?
Bitterness is a taste sensation that is perceived at the back of the tongue and should not be confused with astringency or sourness. The bitter defect in cheese nearly always results from the excessive accumulation of hydrophobic peptides derived from the caseins, particularly from the hydrophobic C-terminal region of ^-casein. Peptides with a molecular mass < ca. 6kDa and a mean hydro-phobicity > 1400 cal per residue are often bitter. Bitterness is a serious problem in low-fat cheeses, probably due to reduced partioning of hydrophobic peptides into the fat phase. Bitterness also develops in cheeses with a low salt level (i.e. low ionic strength) . Low ionic strength weakens hydrophobic interactions between the caseins and facilitates the action of enzymes from the coagulant on hydrophobic regions of the caseins, particularly the C-terminal region of casein, resulting in the excessive production of hydrophobic peptides (particularly ,3-CN f193-209).
The development of bitterness in cheese is due to incorrect patterns of proteolysis causing either the excessive production of bitter peptides (usually by enzymes from the coagulant ) or insufficient peptidase activity to degrade hydrophobic peptides to free amino acids  (Fig. 1).
The following questions should be considered if bitterness develops unexpectedly:
• Does the milk have an excessive psychrotroph count ? If so, heat-stable proteinases may be responsible for the production of bitter peptides.
Fig. 1 Schematic representation of the production and degradation of bitter peptides in cheese during ripening.
Fig. 1 Schematic representation of the production and degradation of bitter peptides in cheese during ripening.
• Has the rennet preparation been changed recently? Have any other proteolytic enzymes (e.g. in preparations used to accelerate ripening) been added to the milk or cheese?
• Has the starter culture been changed? What is the peptidase activity of the starter and what is the specificity on the caseins of its cell envelope-associated proteinase? Are the numbers of starter cells too high or too low?
• Is the NaCl content of the cheese low?
• Has the fat content of the cheese been reduced?
Strategies to ameliorate bitterness in cheese include changing the rennet preparation used to coagulate the milk to one more suitable for the application, using a starter culture or adjunct with high peptidase activity and ensuring an adequate NaCl level in the cheese (but note the effects of varying NaCl levels [43, 46, 47]).
LEMIEUX, L. and SIMARD, R.E. (1991). Bitter flavour in dairy products. I. A review of the factors likely to influence its development, mainly in cheese manufacture. Lait 71, 599-636.
McSweeney, P.L.H. (1997). The flavour of milk and dairy products. Part III. Cheese: taste. Int. J. Dairy Technol. 50, 123-128.
90 What is hydrolytic rancidity and how can it be avoided?
In high-fat foods, lipids may undergo hydrolytic or oxidative degradations. However, the low oxidation-reduction potential of cheese (ca. —250 to —350 mV) and the low levels of polyunsaturated fatty acids in milk fat mean that lipid oxidation occurs to a very limited extent in cheese. Hence, the major pathway for degradation of lipids in cheese is hydrolytic and involves the action of lipases on the triacylglyercols of milkfat to produce free fatty acids (FFA) and partial glycerides.
The lipolytic agents in cheese originate from five principal sources:
1. The milk contains high levels of lipoprotein lipase (LPL). This indigenous enzyme is largely inactivated during pasteurisation  and hence is of significance mainly in varieties made from raw milk.
2. The coagulant may or may not contain lipolytic enzymes . Commercial rennet extracts used to coagulate the milk for the majority of cheese varieties should be free from lipase activity, but rennet pastes used for the manufacture of certain Italian (e.g. the various Pecorino varieties and Provolone) and some traditional Greek cheeses contains a potent lipase, pregastric esterase (PGE). PGE originates from glands underneath the tongue and is washed into the stomach as the animal suckles. Rennet pastes are produced by mascerating the partially dried stomachs of the young of the dairy animal, and their contents, into a paste.
3. The starter and non-starter lactic acid bacteria [18, 56] are generally weakly lipolytic but their intracellular lipase/esterases do contribute to the low levels of lipolysis found in varieties such as Cheddar  and Gouda .
4. Secondary organisms [128, 137, 141, 142] (e.g. the moulds in mould-ripened varieties or the smear organisms in smear cheeses) may be very lipolytic. In particular, Penicillium roqueforti, which develops within Blue cheeses during ripening, produces potent lipases which lead to extensive lipolysis during ripening.
5. Exogenous lipases may be used to accelerate ripening.
Levels of FFA are commonly used as indices of lipolysis and vary considerably between different cheeses. The extent of lipolysis is a characteristic of the ripening of each variety and cheeses with high levels of lipolysis generally have one or more strongly lipolytic agents, are made from raw milk and/or are ripened at elevated temperatures or for long periods of time. Levels of lipolysis typical of Blue cheese (~30 000mgkg—1) or certain hard Italian cheeses (-15 000mgkg—1) would be considered as a serious defect in varieties such as Cheddar or Gouda which are characterised by much more limited levels of lipolysis (-1000-4000 and -400mgkg—1, respectively).
Factors to consider if an undesirable rancid flavour develops during ripening include:
• damage to the milk fat globule membrane prior to pasteurisation which could allow access of active LPL to its substrate ;
• use of raw milk for cheesemaking;
• high numbers of psychrotrophs  in the raw milk which can produce heat-stable lipases;
• ripening at elevated temperatures and/or for prolonged durations;
• changes to the starter used or differences in the non-starter microflora  during ripening;
• possible lipolytic activity in the rennet preparation used ;
• presence of moulds or smear organisms  during ripening;
• lipolytic activity in any enzyme preparations used to accelerate ripening.
COLLINS, Y.F., McSWEENEY, P.L.H. and WILKINSON, M.G. (2004). Lipolysis and catabolism of fatty acids in cheese, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 374-389.
91 What is late gas blowing and how may this defect be avoided?
Late gas blowing is a serious defect associated with certain hard cheeses  caused by the anaerobic fermentation of lactate by Clostridium spp. (particularly C. tryobutyricum) to butyrate, H2 and C02 (Fig. 1). Late gas blowing is principally a defect of brine-salted varieties since diffusion of NaCl through the cheese mass causes a time lag for salt to reach concentrations inhibitory to the growth of C. tyrobutyricum . Because it is not a brine-salted cheese, Cheddar is not very susceptible to late gas blowing.
Late gas blowing can be avoided by minimising the numbers of spores in the milk by good hygiene and avoiding feeding silage to the cows. Germination of spores and the growth of the vegetative cells may be inhibited usually by the use of nitrate or lysozyme. Spores may also be removed from the milk by bactofugation or microfiltration. In general, bactofugation, an increased level of NaCl in the cheese and a reduced ripening temperature are effective measures for reducing gas production by Clostridium spp.
liAD+J NASfl A
Fig. 1 Pathway for the anaerobic metabolism of lactate to butyrate, C02 and H2 by Clostridium tyrobutyricum which causes late gas blowing (from McSweeney and Fox,
2004, with permission).
McSWEENEY, P.L.H. and FOX, p.F. (2004). Metabolism of residual lactose and of lactate and citrate, in Cheese: Chemistry, Physics and Microbiology Volume 1 General Aspects, 3rd edn, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds.), Elsevier Academic Press, Amsterdam, pp. 361-371.
SU, Y.C. and INGHAM, S.C. (2000). Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp. in Gouda cheese. Int. J. Food Microbiol. 54, 147-154.
92 What general factors affect the texture of hard and semi-hard cheeses?
The texture of a ripened hard or semi-hard cheese is determined by a number of factors. The initial composition of the milk [2, 3], the rate and extent of acidification during manufacture  and the degree of heating and moisture removal during manufacture  determine the basic curd structure. This basic curd structure comprises a casein network in which fat globules and moisture are entrapped. Water is both bound to the casein and also fills the interstices of the curd matrix. Texture formation is critically influenced by the relative content of protein, fat and water in this structural network. The biochemical and physicochemical changes that occur in the structure during maturation determine the ultimate texture of the ripened cheese.
The first stage in the manufacturing process which influences cheese texture is the preparation of milk by standardisation of the casein to fat ratio . This determines the fat-in-dry-matter content of the final cheese. The temperature of the standardised milk is adjusted and the milk is acidified using mesophilic or thermophilic cultures . The cultures are carefully selected for the particular cheese variety. This ensures acidification proceeds at the correct rate and solubilisation of colloidal calcium phosphate  is controlled, thereby ensuring the final cheese has the correct composition. The coagulant is added to the milk and a gel is formed . The type and quantity of coagulant are critical: the coagulation temperature, the rate of acid development and the pH of the curd at cutting will determine the coagulant activity and its retention in the curd  and hence the degree of proteolysis during ripening. Once the gel is formed, the curd is cut. The treatment of the cut curd is crucial to ensuring the desired texture is achieved. The size of the curd particles following cutting, the cook or scald temperature, curd washing in which whey is removed and water is added, the pH of the curd at whey drainage, the temperature of the curd at stretching (for Mozzarella ), the extent of cheddaring (for Cheddar ), the method of salting (dry-salting or brining)  and the amount of salt used, all influence cheese texture. The temperature and humidity at which the cheese is stored during ripening can be used to control the cheese microflora , enzymatic activity and texture formation. Proteolysis is the most important biochemical event in cheese ripening  and greatly influences the development of texture. Development of the cheese structure and texture during ripening is primarily achieved by the degradation of the paracasein complex by the proteinases of the coagulant. The duration of the ripening period will determine the extent of proteolysis. However, the softening of texture in the initial stages of ripening results from the solubilisation of colloidal calcium phosphate associated with the paracasein matrix of the cheese  rather than specific chymosin-mediated proteolysis.
GUNASEKARAN, S. and AK, M.M. (2003). Cheese Rheology and Texture, CRC Press, Boca Raton, FL.
O'MAHONY, J., LUCEY, J. and McSWEENEY, P.L.H. (2005). Chymosin mediated proteolysis, calcium solubilisation, and texture development during the ripening of cheddar cheese. J. Dairy Sci. 88, 3101-3114.
93 Cheese is weak bodied. What strategies could be adopted to produce a firmer cheese and what are the effects of each treatment?
A weak-bodied cheese has a weak casein network structure. The main causes of weak-bodied cheese are high levels of fat and moisture compared with casein levels. A weak network structure can be corrected by increasing the compactness of the curd matrix. This can be achieved primarily by reducing the fat and moisture content of the curd. Standardisation of milk  to a higher casein to fat ratio and adjustment of the cheesemaking protocol to enhance moisture loss  (Table 1) will produce substantial improvements in texture. Secondary factors to be considered are increasing the pH at whey drainage to increase calcium levels in curd [4, 17] while decreasing retention of rennet 
Table 1 Adjustments to the cheesemaking protocol to improve texture of a weak-bodied cheese
Standardise milk  to a higher casein to fat ratio
Add calcium chloride to milk 
Increase the amount of starter culture  or prolong the ripening period
Cut the coagulum into smaller cubes
Increase the cooking temperature
Increase the stirring time Increase the pH at whey drainage
Decrease curd size on milling
Increase salt addition at milling
Decrease the ripening temperature
Lowers the FDM in the cheese
Creates a more compact casein network structure
Improved gel formation and syneresis of curd
Slight improvement in whey expulsion
Improved whey expulsion
Improved whey expulsion
Improved whey expulsion Reduces the retention of chymosin and plasmin and increases calcium in curd
Improves whey expulsion Improves whey expulsion
Increases the amount of intact casein
Adjust cheesemaking process to maintain MNFS, S/M and pH
Excess use of calcium chloride will give rise to bitterness during ripening
May increase losses of fat and fines losses
Reduced casein degradation during ripening
S/M levels critical controls the activity of residual rennet and plasmin in cheese
Reduced rate of flavour development possible
FDM = fat-in-dry-matter; MNFS = moisture-in-non-fat-solids; S/M = salt-in-moisture.
and plasmin and increasing salt-in-moisture levels and decreasing ripening temperatures. The salt-in-moisture level in the curd and the ripening temperature control the activity of residual rennet and plasmin in cheese. Increasing the salt-in-moisture level and lowering the ripening temperature can reduce proteolysis during maturation  and result in higher levels of intact casein in cheese which will produce a firmer cheese. Table 1 describes a number of approaches to improve the texture of a weak-bodied cheese.
94 What strategies should be adopted and what are the effects of each treatment to obtain a less acid Cheddar cheese?
Acid flavour is a component of the overall sensory profile of a Cheddar . An excess or imbalance of acid taste may be regarded as a quality defect. The formation and sensory perception of acid flavours are complex issues which remain poorly understood. To produce a less acid cheese, the activity of the starter [17, 18] must be reduced during cheesemaking. This is effectively achieved by reducing the level of starter culture added and adjusting temperatures  and cheesemaking protocol. Cheeses develop acidity during production as lactose is fermented by the starter cultures. When lactose is depleted within 48 h of manufacture, the acidity of Cheddar decreases slightly as the cheese matures. Data from manufacturers indicate that cheeses produced using the same recipes at different manufacturing sites can differ markedly in acidic taste. This implies that additional factors play a role in the acid flavour of cheese. Such factors might include the isomeric forms of lactic acid (D or l) , the presence of other acids (acetic, citric, fatty acids, amino acids), pH (dissociation of acids), buffering capacity , salt level, fat content, degree of proteolysis  and cheese texture.
Development of effective strategies to control acid flavour in cheese requires understanding of not only the technological parameters that contribute to development of acidity but also other factors that influence the sensory perception of acid flavours. The mechanism of perception of acid flavours in cheese is complex and only partially understood. The cheese matrix contains many components that contribute to the flavour profile and potentially affect the perception of acidity. In a recent survey of the sensory character of retail Cheddars in the UK, interactive effects were noted in perception of acid and creamy flavours (Fig. 1). This effect was consistent in mild, mature, vintage Cheddars and half-fat Cheddars. Acid flavours were lowest in the mild category cheeses and had a tendency to increase with maturity but there was much variability in perception of acid flavours in each category.
95 What strategies can be adopted to soften the texture of a hard cheese?
The major structure-forming constituent in cheese is the casein matrix in which fat globules are entrapped. Water or serum is both bound to the casein micelles and fills the interstices of the matrix. The network structure is influenced by the relative content of protein, fat and moisture, as well as the biochemical activities that occur during maturation. An overfirm cheese has an excessively compact casein network structure which is generally associated with reduced fat and moisture levels in curd.
Increasing the fat and water content of the matrix opens up the protein structure and softens the texture. Strategies for adjusting fat and moisture levels are shown in Table 1. Removing fat has a greater effect on texture than removing moisture. In low-fat Cheddar, the casein matrix is extremely compact and the texture is overfirm even when the moisture content is substantially
Table 1 Process modifications to soften the texture of an overfirm cheese
Standardise milk to a lower casein-to-fat ratio 
Decrease the amount of starter culture  or shorten the ripening period
Cut the coagulum into larger cubes
Decrease the cooking temperature  Decrease the stirring time
Decrease the pH at whey drainage
Increase curd size on milling Decrease salt addition at milling
Increase the ripening temperature
Increases the FDM in the cheese
Creates a more open casein network structure
Slight reduction in whey expulsion
Reduction in whey expulsion Reduction in whey expulsion
Reduction in whey expulsion
Increase the retention of chymosin and plasmin in curd Calcium retention in curd decreased
Reduction in whey expulsion Reduction in whey expulsion
Enhanced degradation of casein 
Adjust the cheesemaking process to enhance MNFS
May reduce losses of fat and fines
Increased casein degradation during ripening, giving softer texture
S/M levels critical. Controls the activity of residual rennet and plasmin in cheese
Off-flavours if relative rates of proteinase and peptidase activity unbalanced 
FDM = fat-in-dry-matter; MNFS = moisture-in-non-fat-solids; S/M = salt-in-moisture.
increased . To produce a softer cheese, the casein to fat ratio in milk for manufacture should be decreased to increase the fat-in-dry-matter level in the curd. Cheesemaking parameters should then be adjusted to enhance moisture retention. Increasing the proportion of unsaturated fats in cheese would also result in a softer cheese. The addition of water binders, e.g. denatured whey proteins, generally improves the texture of low-fat cheeses.
To enhance proteolysis during ripening so that casein matrix is broken down and texture weakened, the cheesemaker should decrease the pH at which the whey is drained from the curd. This decreases the calcium levels in cheese and increases the residual levels of chymosin and plasmin [4, 28]. The salt-in-moisture level should be reduced and the maturation temperature increased to accelerate proteolysis.
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