17 Technology of cheese manufacture III: Cheese ripening including accelerated ripening

Atanu Jana

Ripening of cheese

All cheeses do not require ripening; these include cottage, cream, quark, etc. Several cheeses are required to be ripened under controlled conditions of temperature and/or relative humidity in order to develop distinct attributes (viz., flavor, body and texture) in the resultant aged cheese. Examples are cheese that require ripening include Cheddar, Swiss, Edam, Gouda, Roquefort, Blue, etc. Most rennet coagulated cheeses are ripened after manufacture for periods ranging from 2 weeks (e.g. Mozzarella) to more than a year (e.g. Parmigiano-Reggiano or extra-mature Cheddar). Cheese flavour is extremely complex and “Component Balance Theory” proposes that cheese flavour is due to the interaction, in correct proportions, of many compounds, present at specific concentrations.

Cheese ripening involves a concerted series of microbiological, chemical and biochemical reactions (i.e. glycolysis, lipolysis and proteolysis) that are ultimately responsible for the development of characteristic texture, flavour, aroma and appearance of individual cheese varieties. Cheese ripening, basically involves the catabolism of major milk components including protein, fat, and lactose. Catabolism is brought about by various enzymes which come from sources like, milk, lactic acid bacteria (LAB), coagulant, non-starter LAB (NSLAB), and secondary starters.

The relative importance of each of the biochemical processes involved in cheese ripening depends on the variety of cheese; lipolysis is important in mold ripened cheese like Blue/Roquefort, while proteolysis is of significance in Cheddar cheese.

Factors affecting cheese ripening

The primary factors that influence cheese ripening are summarized herein:

1. Storage temperature and humidity, humidity being less important for cheeses hermetically packed (e.g., with wax coating).
2. Chemical composition of the curd especially fat content, level of amino acids, fatty acids, and other by-products of enzymatic action.
3. Residual microflora of the curd, primarily those used as starter culture. The cheese maker can do little to influence it except in the case of blue-veined or surface-ripened cheeses.

Temperature and humidity are factors that cheese makers can control during ripening. In general, higher temperatures favour microbial growth and other biochemical reactions occurring in the curd. Thus, cheeses matured at different temperatures can have different flavor profiles.

Ripening conditions

Swiss-type Emmental is held at a low temperature initially (10 to 15°C) to facilitate the growth of LAB. Later, the temperature is raised (20 to 24°C) so that propionic bacteria can grow. These are essential for the characteristic Emmental flavor and „eyes‟. For blue-veined cheeses (e.g., Gorgonzola, Roquefort, Stilton), warm-temperature storage is followed by low-temperature storage.

Prevailing humidity during storage (80 to 85 % RH) helps to control the moisture content of cheeses without moisture barriers (i.e. coatings). In general, higher moisture content promotes growth of microorganisms. In addition to temperature and moisture, other factors like curd pH, inhibitory substances (e.g. antibodies, salts), and oxidation–reduction potential affect the microbial population in the cheese. The enzymes relevant for maturation in most hard cheeses are active in the pH range of 4.9 to 5.5, and in soft cheeses from pH 5.3 to 6.0.

Changes taking place during cheese ripening

Lactic acid is an important precursor for the production of other volatile flavor compounds during ripening such as fatty acid esters, ethyl esters, thioesters, fatty acid lactones – the principal class of flavor compounds. Blue cheese contains n-methyl ketones (alkan-2-ones).

I. Proteolysis during cheese ripening

Proteolysis is the most complex process, the extent of which depends on the variety i.e. from very limited in mozzarella to very extensive in blue-mould cheeses. Proteolysis is the most significant primary ripening process in cheese, having an impact on both texture and flavour of cheese. The general reaction steps are: Initial hydrolysis of caseins by residual coagulant and plasmin to large peptides.Breakdown of large peptides by starter proteinases and peptidases into medium and small peptides.  Further hydrolysis of the medium and small peptides by starter peptidases into dipeptides, tripeptides and free amino acids.

Proteolysis contributes to the development of cheese texture, through hydrolysis of the protein matrix and an increase in the pH due to liberation of ammonia from amino acid. Proteolysis takes place in two phases viz., primary and secondary. Primary proteolysis represents the extent of breakdown of the native casein. Secondary proteolysis is further degradation leading to formation of peptides and free amino acids. Proteolysis in cheese during ripening is catalyzed by proteinases and peptides from six sources viz., the coagulant, milk, starter LAB, NSLAB, secondary starter and exogenous proteinases and peptidases.

While the level of residual coagulant in cheese is dependent on the pH of milk at setting, the plasmin activity is dependent on the cooking temperature used during manufacture, being higher in certain cheese types like Swiss cheese as against that of Cheddar cheese. The peptides produced by the action of residual coagulant and plasmin are often either tasteless or bitter and do not contribute directly to the typical taste of cheese. However, the mixture of small peptides and amino acids directly influences the taste and mouth feel of cheese. The free amino acids may be further catabolized into flavour compounds that are unique for each cheese type and are dependent on the types of enzymes and microorganisms (particularly NSLAB) present in cheese. Microbiological changes include death of the starter culture and the growth of non-starter flora and in certain cheeses, the growth of a secondary microflora.

II.    Lipolysis during cheese ripening

The level of lipolysis is limited in Swiss-type and semi-hard cheeses, whereas it is extensive in case of hard Italian mold ripened (5–20 %), blue veined (18–25 %), Romano, Provolone and goat milk cheeses. Free fatty acids (FFAs) are important precursors for several volatile compounds which contribute to the flavor of cheese. FFAs with C4-C10 are strongly flavored. Lipases in cheese originate from milk, rennet paste, starter bacteria, secondary starter microorganisms, NSLAB and exogenous lipase preparations. LAB are weakly lipolytic, but their enzymes contribute to lipolysis in Cheddar cheese at low level. Propionic acid bacteria (used in Swiss cheese) are more lipolytic than LAB and hydrolyze triglycerides at maximum rate to tripropionin, tributyrin, tricaproin and subsequently caprylin. Pencillium roqueforti (mold) produces potent extracellular lipases which are responsible for extensive lipolysis observed in blue cheese (mold ripened). FFAs are precursors of flavour and aroma compounds, such as methyl ketones, lactones, esters, alkanes and secondary alcohols. Methyl ketones are formed in blue cheese by the action of P. roqueforti.

In case of Cheddar cheese, lipolysis results in formation of FFAs, which are flavouring constituents of cheese and can be precursors of flavor compounds like methyl ketones, alcohols and lactones. Bacterial ripened varieties (Edam, Swiss, Cheddar) have low levels of FFA i.e. 200-1000 mg/kg, while blue cheese has very high levels of FFA i.e. 30,000 mg/kg. The total FFAs in Emmental cheese is reported to be around 6,600 mg/kg.

III. Glycolysis during cheese ripening

Cheese curd contains low level of residual lactose which is metabolized via a range of pathways. In glycolysis, lactose is converted to pyruvate to lactate; latter is catalyzed by lactate dehydrogenase. Lactate is an important substrate for a range of reactions which contribute to cheese ripening by metabolism to acetate and CO2 by NSLAB flora through oxidative pathway. The resultant lactic acid may be catabolized to other compounds, e.g. CO2 and water by the surface mould in Camembert, or to propionic acid, acetic acid and CO2 in Emmental type cheese. The development of eye in Swiss cheese is due to production of CO2 and water by Propionibacterium freudenreichii subsp. shermanii by metabolizing lactate in cheese curd.

IV. Citrate metabolism

Citrate is metabolized by Streptococcus diacetylactis, Leuconostoc mesenteroides [iods] subsp. cremoris to diacetyl, acetate, acetoin and CO2. However, it is not metabolized by Streptococcus thermophilus or thermophilic lactobacilli. Diacetyl contributes to the flavour of cottage cheese.

Accelerated ripening of cheese

Cheese ripening occurs slowly, thereby making the process an expensive one. Ripening of hard cheese varieties is a long and costly process because of capital immobilization, large refrigerated storage facilities, weight losses, and spoilage caused by undesirable fermentations. For instance Parmesan and extra-mature Cheddar are ripened for at least 18 months. Ripening is still not controllable precisely, i.e. the quality and intensity of flavor cannot be predicted precisely. Therefore, there is an economic incentive for the development of methods for accelerating cheese ripening.

Cheese (especially hard) derived from buffalo milk takes more time to ripen compared to use of cow‟s milk. Ripening of buffaloes’ milk Cheddar cheese could be accelerated by addition of Lactobacillus casei or a lipase + proteinase preparation in cheese manufacture; the cheese matured in 6 months as against 10 months without adopting such acceleration method.

Traditionally, cheese varieties are ripened at a characteristic temperature which is chosen to suit the secondary microflora, e.g. Propionibacterium or molds. Temperatures of 8-12oC are common, exception being Swiss-type cheeses that are exposed at ~20oC for a period of 3-4 weeks to induce growth of Propionibacterium (for eye formation and flavor). Cheddar is ripened at ~ 15oC, but lately it is being practiced at 6-8oC, reducing the risk of off-flavour development.

Of the three primary events in cheese ripening, i.e., glycolysis, lipolysis, and proteolysis, the latter one is usually the rate-limiting one. Glycolysis is normally very rapid and is complete in most varieties within 24 hr. The modification and catabolism of lactate is either of little or no consequence (e.g., Cheddar or Dutch varieties) or is quite rapid i.e. 2-3 weeks (e.g., Swiss and Camembert types). Lipolysis is limited in most cheese varieties, exceptions being some Italian varieties, e.g., Romano and Provolone, and blue varieties.

The intensity of the flavor of several varieties, including Cheddar, can be increased by adding exogenous lipases, but such practices are not used commercially. Acceleration of cheese ripening is focused on proteolysis, especially in hard, low-moisture varieties, in particular Cheddar. Low-fat cheeses have attracted much attention recently. Another area of interest is the production of cheese-like products e.g., enzyme modified cheeses (EMC), for use in the preparation of food products (i.e. processed cheeses, cheese sauces, cheese dips).

Acceleration in cheese ripening has been attempted through use of:

Elevation of ripening temperature (13oC/24 weeks)  Increasing the numbers of viable cells with selective attributes Using cheese slurry systems

Addition of crude cell-free extracts or partially purified extracts to the cheese milk or curd  Use of exogenous enzymes and entrapped enzymes Attenuated bacterial cells   Culture adjunct microorganisms   Specifically designed cultures of LAB with desired attributes of proteolytic enzymatic activity   Physical methods

Means to accelerate cheese ripening

Exogenous enzymes: Commercial proteinase enzymes such as Neutrase (neutral proteinase from Bacillus subtilis). Flavourzyme, Accelase AM317, Accelase AHC50, Accelerzyme CPG has been utilized to accelerate cheese ripening.

The proteolytic enzymes are mixed with salt and added to the milled Cheddar curd just prior to moulding in order to accelerate ripening. The microcapsules are added to the cheese milk, which gets entrapped in the curd, thus ensuring uniform distribution in the curd and preventing its loss in whey. The microcapsules disintegrate during cooking or ripening, releasing the entrapped enzyme into the cheese matrix. Liposomes are the preferred types of capsules. An acceptable cheese was obtained by accelerated ripening using 0.025% of each enzyme (proteolytic + lipolytic enzymes, 1:1 ratio) with 5 % of a starter culture and ripening at 20oC for 12 days. Such a cheese base could be incorporated into processed cheese products at levels of 10-20 %, to obtain an appropriate flavour. Use of Aeromonas caviae T-64 aminopeptidase along with neutrase enzyme helped in accelerating Gouda cheese ripening; intense cheese flavor was obtained in just 8-12 weeks of ripening.

Enzyme (b-galactosidase, proteinases, lipases) may be added to the cheese milk, curd, pressed curd, or even in encapsulated form. Lipases and proteinases encapsulated in κ-carrageenan, when added to cheese milk together with rennet, accelerated the proteolysis as well as lipolysis in Kasher cheese. Proteolytic enzymes encapsulated in alginate polymers have been used to enable the enzymes to release from the capsule during ripening of cheese. Enzymes encapsulated in liposomes has been successfully used to accelerate Cheddar cheese ripening; the recommended dosage of bacterial proteinase was up to 1 x 10–5 AU/g cheese curd and up to 2 x 10–6 AU/g cheese curd of fungal proteinase. Use of 0.5 g KCl + 0.03 g MnCl2/kg milk and 1.35 g proteinase+   0.65 g triacylglycerol lipase/kg curd is recommended for reducing the ripening time of Ras cheese.

Adjunct microorganisms: Use of adjuncts microorganisms, over and above the normal starter, such as lactase-negative mutants, mutants produced by UV or ionizing radiation, heat- or freeze-shocked cultures, and lyzed cells, cell walls or cell-free extracts added to cheese milk or curd has been employed to accelerate cheese ripening. Starters modified by physical, chemical or genetic means can very well be adopted. Intracellular cell-free enzyme extracts (CFE) of the cheese starter Streptococcus lactis NCDO 712 when added to curd at 1.5-7.5 mg/g curd enhanced the flavour-accelerating effect (through rapid release of peptide and amino acid nitrogen) of a commercial neutral proteinase viz., Neutrase – proteinase from Bacillus subtilis (Novo), without increasing the gross proteolysis in Cheddar cheese. Current trend are to combine proteinase with peptidases.

Use of yeast Debaryomyces hansenii and Yarrowia lipolyticaare, known for their proteolytic and lipolytic activity, as adjuncts in Cheddar cheese processing led to accelerated ripening and flavor development.

Increased starter cell numbers: Increasing potential starter cell number has been made possible without increased acid development in cheese, either by use of lactase-negative (Lac-) or attenuated starters in order to accelerate ripening. Attenuated starter can be prepared by heat-shocking or freeze-shocking Lactococcus or Lactobacillus cells such that their acid-producing ability is destroyed, but much of their proteolytic and peptidolytic activities are retained.

Lyzable starter strains: Another approach is to select or develop starter strains that lyze and release their intracellular enzymes quickly in the early stages of ripening. This required induction with the food grade peptide nisin which led to expression of genes encoding lytic proteins in Lactococcus lactis. Fast lyzing strains of Lactococcus have been reported to give faster rates of ripening in case of Cheddar and Saint Paulin cheeses.

Cheese slurries: Flavor has been reported to develop very rapidly (1 week) in cheese slurries containing about 40 % solids. Cheese slurries can be incorporated into cheese milk, or to the curd before cheddaring or to the salted curd before pressing.

Advantages and disadvantages of accelerating cheese ripening

Merits of accelerated ripening

Cheese ripening is an expensive (about $50 per ton per month for ripening cheese) process. Hence, technologies for reducing the time and cost for storing and maturing cheese until it is sold have economic significance to the dairy industry and cheese consumers.

Accelerated ripening techniques can also be used to manufacture products such as EMC for use in processed cheese or as ingredients in products in which high cheese flavour is required.

Demerits of accelerated ripening

Addition of enzymes, e.g. b-galactosidase, proteinases (mainly of microbial origin) and lipases, either to cheese milk or to the curd, allows good control of flavour and aroma but may lead to over-ripening and alteration of cheese texture.

Bitterness in cheese (hard varieties) is one possible defect observed in cheese subjected to accelerated ripening. However, such problem can be tackled through use of debittering peptidases.

Indices of cheese maturation

It is desirable to monitor the ripening changes so that the cheese can be marketed at optimal stage of cheese ripening. The several methods that are used to monitor indices of cheese maturation are mentioned below.

Physical indices – Cheese rheology

Chemical indices of cheese maturation

1. Monitoring proteolysis

• Soluble nitrogen
• Trichloro acetic acid (TCA) soluble nitrogen
• Measuring liberated amino acids viz., free L-glutamic acid, Tyrosine value
• Liberated amino groups (Amino-N)
• Electrophoresis of cheese

„Ripening index‟ is usually obtained as ratio of Water soluble nitrogen to total nitrogen in cheese.

1. Monitoring lipolysis

• Acid Degree Value
• Thio Barbituric Acid value
• Total Volatile Fatty Acids

Web links

• http://cheeseforum.org/forum/index.php?action=dlattach;topic=6939.0