Microbial changes in Camembert

In Camembert and related surface mould-ripened cheeses, the mesophilic starter reaches perhaps 10^9 cfu/g at the end of manufacture. Spores of Penicillium camemberti may be added to the milk or sprayed on the surface of the cheese after moulding. Initially, the surface microflora is composed of adventitious acid-tolerant yeasts and Geotrichum candidum. P. camemberti appears after about 6 days and dominates the ripening of Camemert and Brie-type cheeses. Eventually, towards the end of ripening, a Gram-positive bacterial microflora begins to develop. These organisms, that are often pigmented, are adventitious and similar to those found of the surface of smear-ripened cheeses.

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Fate of the starter in Cheddar

The starter grows during Cheddar cheese manufacture from ~10^7 to 10^8-10^9 cfu/g and reduces the pH from that of milk (~6.7) to ~5.4 at the point of milling. Since Cheddar is a dry-salted variety, and unlike many other cheeses, acidification must be close to complete at the end of manufacture and before salt addition. Salt-in-moisture levels increase rapidly in Cheddar due to the large surface area of the curd chips.

During the early stages of ripening, viable starter counts decrease rapidly at a strain-dependent rate. This decline is due to the unfavourable conditions in cheese for the growth and survival of lactococci: low pH, high concentration of NaCl and lack of fermentable carbohydrate. The salt-in-moisture level largely determines the rate of utilisation of residual lactose in the cheese which is of significance to cheese quality.

After death, the lactococci lyse at a rate that is strain dependent and contribute many important enzymes to cheese ripening (particularly its battery of peptidases but also esterases). There is evidence emerging that starter cells may be metabolically active but non-culturable during ripening and that they may contribute to amino acid catabolism in that state.

NSLAB

Non-starter lactic acid bacteria (NSLAB) are a common component of the microflora of many cheeses and nearly all hard ripened varieties. In a cheese such as Cheddar, they are perhaps the only aspect of the cheese that remains largely uncontrolled.

NSLAB in Cheddar are usually wild strains of Lactobacillus paracasei/Lb. casei that probably gain access to the cheese from the raw milk by surviving pasteurization or from the cheesemaking environment. These organisms grow from very low numbers and typically reach about 10^7-10^8 cfu/g within 2-3 months. They have enzyme systems generally similar to those of Lactococcus and probably contribute to ripening or indeed to the development of off-flavours (research in New Zealand has suggested many flavour defects in Cheddar made under best practice are due to NSLAB). NSLAB are the dominant viable microflora of mature Cheddar cheese. Ripening temperature and, significantly, the rate of cooling of Cheddar blocks after manufacture are major factors which control the growth rate of NSLAB.

Techniques in the literature used to study the contribution of NSLAB to ripening include comparison of raw and pasteurised milk cheeses, microfiltration, use of antibiotics, aseptic cheesemaking and low ripening temperatures.

Further information:

Fox, P.F., P.L.H. McSweeney and C.M. Lynch (1998). Significance of non-starter lactic acid bacteria in Cheddar cheese. Australian Journal of Dairy Technology 53, 83-89.

Enzyme assay kits

Enzyme assay kits are convenient ways to measure certain constitutents of cheese (e.g., lactose, D/L-lactate or citrate). The the example shown below, D-lactate is determined by using D-lactate dehydrogenase which catalyses the conversion of D-lactate to pyruvate and NAD+ to NADH. This reaction does not naturally go to completion so a second enzyme, glutamate-pyruvate transaminase, which reacts pyruvate and glutamate, is used to pull the first reaction to completion. The conversion of NAD+ to NADH is measured spectrophotometrically as the oxidized and reduced form of this co-factor absorb differently at 340 nm.

Enzyme assay kits are quick and convenient as all reagents necessary come with the kit. However, their use can be expensive for large number of samples and the kits cannot be stored for long periods of time without loss of enzyme activity.

Dairy Technology Expertise in Ireland

The third technology and expertise alert has been prepared for the dairy sector by Relay. This highlights the technology and expertise as well as the facilities, equipment, services and key contacts at Irish institutes and universities. It is hoped that this document will help researchers quickly identify who can help with R&D/technical challenges and where the relevant expertise and equipment is available.

The Department of Agriculture, Fisheries and Food through the FIRM programme has funded 104 projects directly aimed at supporting the dairy industry and this research has contributed in a significant way to the expertise at the institutes and universities.

To download this document, please click here: Dairy Technology and Expertise in Ireland.

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alpha-Keto acids. 2

In addition to conversion to hydroxyacids by the action of 2-hydroxyacid dehydrogenases, alpha-keto acids can be decarboxylated to the corresponding aldehyde. In the example of the degradation of tryosine, its alpha-keto acid (p-hydroxy phenylpyruvate) is converted to phydroxy phenylethanal, which in turn can be oxidised to the corresponding alcohol (p-hydroxy phenylethanol) or reduced to the corresponding carboxylic acid (p-hydroxy phenylethanoic acid). alpha-Keto acids can also undergo a series of chemical degradations leading, in the example of tyrosine to products such as p-hydroxy benzaldehyde.

alpha-Keto acids. 1

The alpha-keto acids produced by aminotransferase activity are relatively unstable and do not accumulate in cheese but are rather degraded via a range of pathways. Taking tyrosine as an example, its alpha-keto acid (p-hydroxy phenylpyruvate) can be degraded by 2-hydroxyacid dehydrogenases to the corresponding hydroxy acid (p-hydroxy phenyl lactate). Other pathways of degradation of alpha-keto acids include decarboxylations and chemical degradations forming other volatile flavour compounds, which will be discussed in future posts.

Aminotransferases

The catabolism of amino acids to a wide range of volatile flavour compounds is amongst the most important series of reactions in the development of cheese flavour. The key enzymes in the degradation of free amino acids appear to be aminotransferases (ATases) from lactic acid bacteria. ATases are intracellular enzymes whose physiological role is in the interconversion of amino acids. These enzymes require pyridoxal-5'-phosphate (PLP) as a co-factor and catalyse the transfer of the amino group of a donor amino acid (leucine in the example below) to an acceptor molecule, usually alpha-ketoglutarate, forming a product alpha-keto acid corresponding to the donor amino acid (alpha-ketoisocaproate in this example) and glutamic acid. The catalytic mechanism of ATases involves two steps: firstly, the amino group of the donor amino acid is transferred to PLP to yield the product alpha-keto acid and enzyme-bound pyridoxamine-5'-phosphate. Secondly, the amino group is transferred from pyridoxamine-5'-phosphate to the acceptor alpha-keto acid, thus regenerating PLP. The alpha-keto acids formed by ATase action are unstable and degrade to a wide range of compounds via enzymatic and/or chemical pathways.

Syneresis- V

Stirring the curds-whey mixture facilitates heat transfer during cooking, prevents the curd pieces from fusing and promotes syneresis by encouraging collisions with other curd pieces and the vat wall. It is important to stir gently after cutting to avoid curd shattering (and thus yield losses); indeed, some cheesemakers leave a 5-10 min "healing time" after cutting before starting to stir.

In Cheddar-type cheeses, the curds-whey mixture is stirred and cooked to a desired pH (e.g., 6.1-6.2) but in many varieties (e.g., Emmental), the whey is drained at a target temperature. In a few varieties (e.g., stirred-curd Cheddar or Colby cheese), it is normal to stir the curd pieces after drainage ("dry stirring") which also promotes syneresis.

Note: Figure shows the effect of stirring (solid curves) and no stirring (lower broken line) on percentage syneresis as a function of time afer cutting.

Syneresis- IV

Cooking temperature is a major factor that determines the rate of syneresis. Cooking temperature varies from ~31C (Camembert) to 52-55C (Emmental or Parmigiano-Reggiano) and temperature must match the starter. Acid production by lactococci is slowed ~35C and many strains are killed >40C (which is very close to the Cheddar cooking temperature of ~38.5C). Thermophilic starters, while surviving high temperatures do not gro >~52C so syneresis in Swiss cheese (cooked 54-55C) is mainly due to heat; starter grows as curd cools.

Cooking is normally achieved using a jacketed vat although "curd washing" (removal of perhaps 30-40% of the whey and replacement with hot water) is used in Dutch-type cheeses. In addition to increasing the temperature, curd washing reduces the lactose levels and helps to control the final pH of the cheese.

The rate of cooking is important. If it is too fast in the early stages, case hardening can result.

Syneresis- III

Processing variables that influence syneresis include the size of the curd particles. The smaller the pieces, the greater the greater the surface area for whey expulsion hence the greater the syneresis. Indeed, curd for high moisture cheeses not cut but scooped into mould.

Acidification also has a major influence on syneresis. The lower the pH, the greater is syneresis.

Other processing variables that influence syneresis will be discussed in future posts.

Syneresis- II

Factors affecting syneresis are associated with the milk or with the processing operations. Factors that affect syneresis associated with the milk include:

• % Fat. Increasing fat decreases syneresis as fat globules inhibit the movement of moisture to the surface of the curd piece. Increasing the fat content of milk increases cheese yield (Ya, actual yield) by 1.2 x mass of added fat due to increased retention of moisture.


• % Casein. Casein is the structural element in the reticulum of the curd and so increasing casein content of milk results in better syneresis.


• pH of milk. Reducing the pH of milk improves syneresis which is optimal at the isoelectric point of the caseins (pH 4.6). As pH moves towards 4.6 the net charge on the casein is reduced facilitating their interaction.


• Ca2+ generally improves syneresis.


• NaCl added to milk. At low levels, salt added to the milk improves syneresis but at higher levels it reduces syneresis. Addition of salt to milk is a practice used only in the manufacture of a very small number of varieties (e.g., Egyptian Domiati).
  

However, in general processing operations have a greater effect on syneresis than milk composition and will be discussed in future posts.

Syneresis- I

Rennet-induced milk gels are relatively stable if left undisturbed. However, if the gel is cut, broken or exposed to pressure, the para-casein matrix contracts on itself expressing its aqueous phase (as whey) in a process known as syneresis. Controlling syneresis is the key to cheesemaking as it allows the cheesemaker to control moisture which, in turn, largely determines the quality, ripening and stability of the cheese. After the gel has formed, it is subjected to various treatments (cutting, cooking, stirring, acidification, pressing...) to encourage the expulsion of whey.

In hard cheeses such as Swiss and Cheddar syneresis occurs mainly in the vat while the gel for soft (high moisture) varieties such as Camembert may be scooped directly into moulds where whey expulsion is driven mainly by the decrease in pH.

Perhaps surprisingly for such an important parameter, methodology for measuring synersis is relatively poor. Approaches used have included measuring the volume of whey produced or the volume, moisture content or conductivity of the curd. Tracer/marker methods have also been used and some authors mimic the cheesemaking protocol (e.g., addition of starter, cooking) when measuring syneresis. However, all methods have inherent drawbacks.

Processed cheese

Processed cheese (or process cheese) is produced by comminuting, blending and melting one or more natural cheeses, and sometimes optional ingredients, into a smooth homogeneous blend with the aid of heat, mechanical shear and emulsifying salts (often sodium salts of citric or phosphoric acids). Processed cheese is sometimes moulded into blocks or the molten mass of cheese may be cooled on the surface of a large drum, cut into ribbons, interleaved with packaging at cut into slices.

A significant quantity of processed cheese is made worldwide and the product has some advantages over natural cheese including:

• Great variety in flavour, consistency, functionality (e.g., sliceability, consistency, flowability) and consumer appeal.


• Providing an outlet for lower grade natural cheese. This, together with the use of cheap non-cheese ingredients, reduces their cost compared to natural cheese.
  
• Adaptability to the fast-food trade (e.g., use in cheeseburgers).


• Relatively long shelf-life. Processed cheese is relatively stable due to the high temperatures used in processing which inactivate many microorganisms and enzymes.

However, typically processed cheese has a bland flavour.

Further reading:

Guinee, T.P., Caric, M. and Kalab, M. (2004). Pasteurized processed cheese and substitute/imitation cheese products. In Cheese: Chemistry, Physics and Microbiology, Volume 2. Major Cheese Groups, 2nd Edn., P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds), Elsevier, Amsterdam, pp. 349-394.

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Finishing operations-I

Cheese curds may be moulded either after acidification (e.g., Cheddar) or directly after cooking (e.g., Emmental). Curds of high moisture varieties mat together under their own weight but curds of low moisture cheeses must be pressed to form a homogeneous mass. The moulds used give cheese the size and shape characteristic of the variety. Curds (particularly those of low moisture cheeses) should be warm to ensure that they mat together, avoiding mechanical openings which are a defect in most varieties.

Salting-VI. Attainment of equilibrium

Salt absorption is a relatively rapid event taking perhaps 15-30 min for curd chips of Cheddar cheese, ~7.5 h for Camembert and perhaps 15 d for Parmigiano-Reggiano. After NaCl is absorbed into cheese curd, there then must be established a salt equilibrium across the cheese mass.

In dry-salted cheeses, true equilibrium is rarely is ever fully achieved and substantial intra- and inter-block variation in NaCl distribution are common for Cheddar. NaCl equilibrium is established quickly within each curd chip but there is poor transfer of NaCl across the chip boundary. In brine-salted cheeses, however, there is initially a very large NaCl gradient from the outer portion of the cheese towards its centre; attainment of equilibrium is very slow indeed, taking up to 10 months for a large wheel of Parmigiano-Reggiano.

Further reading:

Guinee, T.P. and Fox, P.F. (2004). Salt in cheese: physical, chemical and biological aspects, in Cheese: Chemistry, Physics and Microbiology, Vol. 1 General Aspects, 3rd edition, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds), Elsevier, Amsterdam, pp. 207-259.

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Salting-V. Brine salting

In brine salted cheeses, the difference in osmotic pressure between the (often saturated) brine and the cheese aqueous phase is the driving force for NaCl migration. Diffusion of brine into cheese is an impeded diffusion process (Na+ and Cl- ions must migrate around fat globules and the hydrated casein matrix of cheese).

Factors that affect NaCl uptake in brine-salted cheeses include:

• Concentration gradient. Uptake increases as brine concentration increases from 5-25% (w/w) NaCl.


• Salting time. Uptake increases but at a diminishing rate with salting time.


• Brine temperature. Uptake increases as brine temperature increases from 5 to 20 C. There is a minimum temperature for uptake at ~32C as fat exudes at the cheese surface (above this temperature fat is more liquid and is lost more easily).


• Surface area:volume ratio of the cheeses. Salt uptake increases with increasing surface area:volume ratio.


• Shape of cheese. Assuming an equal surface area:volume ratio, rectangular cheeses brine more quickly than cylindrical cheeses, which in turn brine more quickly that spherical cheeses. (Faster brining is caused in this case by the presence of corners in the cheese which allow brine diffusion from two or more directions.)

• Moisture content of the curd. Rate of NaCl uptake increases as curd moisture increases.


• Fat content of the curd. Increasing fat tends to impede diffusion.


• Curd pH. Uptake decreases as pH increases from 4.7 to 5.7. This effect is caused by increased charge on the caseins as one moves away from their isoelectric point causing greater hydration of the protein and thus tending to impede diffusion.

Further reading:

Guinee, T.P. and Fox, P.F. (2004). Salt in cheese: physical, chemical and biological aspects, in Cheese: Chemistry, Physics and Microbiology, Vol. 1 General Aspects, 3rd edition, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds), Elsevier, Amsterdam, pp. 207-259.

Guinee, T.P. (2007). What factors affect salt uptake in cheese curd? In Cheese Problems Solved, P.L.H. McSweeney (ed), Woodhead, Cambridge, pp. 87-89.

Photo shows an industrial brine bath used to brine Mozzarella-type cheeses moulded into fist-sized pieces.

Salting-IV. Dry salting

Relatively few cheese varieties are dry salted but a high percentage of the world's cheese production is dry salted! This discrepancy can be explained by the fact that Cheddar, the variety produced in the largest quantity, is a dry-salted cheese. In dry salting, the curd is milled (cut) into small pieces at the end of acidification and dry NaCl is sprinkled on the surface of the curd chips.

NaCl dissolves in whey on the surface of curd chips and diffuses inwards (in effect, each individual curd chips is "brined" in its own salty whey) and each curd chip acts as a "mini-cheese". The milled curd chips have a very large surface area: volume ratio which leads to very rapid uptake of salt in Cheddar and related varieties. As high salt-in-moisture inhibits the growth of starter organisms, it is important that the curd has achieved close to its final pH before dry salting.

Factors affecting salt uptake in dry-salted cheese include:

• Quantity of salt added to milled curd: Salt uptake increases as salting level increases)
  
• Mellowing time: Salt uptake increases as mixing time increases from 20 s to 6 min.
  
• Holding time between salt addition/mixing and pressing: Uptake increases as holding time increases.
  
• Curd temperature: Uptake decreases as curd temperature increases from 24 to 41C (this effect is largely due to increased liquefaction of milkfat at the surface of the curd chips which inhibits NaCl uptake).
  
• Surface area: volume ratio of curd chip: NaCl uptake increases as surface area is increased (i.e., by reducing chip size).
  
• Curd moisture: NaCl uptake decreases as curd moisture increases. As moisture increases, there is more syneresis of press whey which removes more NaCl from the chip.
  
• Curd acidity: Salt uptake decreases as acidity decreases (i.e., lower uptake at higher pH).
  

Further reading:

Guinee, T.P. and Fox, P.F. (2004). Salt in cheese: physical, chemical and biological aspects, in Cheese: Chemistry, Physics and Microbiology, Vol. 1 General Aspects, 3rd edition, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds), Elsevier, Amsterdam, pp. 207-259.

Guinee, T.P. (2007). What factors affect salt uptake in cheese curd? In Cheese Problems Solved, P.L.H. McSweeney (ed), Woodhead, Cambridge, pp. 87-89.

Salting-III. How is cheese salted?

There are three basic methods by which cheese is salted. The large majority of cheese varieties worldwide are brine salted. This simply means placing the cheese after forming into a concentrated (and often saturated) NaCl solution which diffuses into the cheese. Cheddar and related varieties are dry salted. After acidification, the curd is milled (cut) into small pieces and dry salt is added and mixed with the curd; the salted curd is then pressed. Some Blue cheese varieties can be salted differently by the repeated surface application of dry salt. After forming, dry NaCl is patted onto the surface of the cheese and this process is repeated as necessary to give the desired salt concentration in the cheese. Different methods of salting have very different effects in cheese as will be discussed later.

The photo above shows milled Cheddar curd being dry salted by students a few years ago in the UCC Pilot Plant dairy. The photo below is of an industrial brine bath taken in a Mozzarella factory in northern Italy.

Salting-II. Salt and bitterness

Chymosin cleaves beta-casein mainly towards its hydrophobic C-terminus. After the Phe-Met bond of kappa-casein, the Leu192-Tyr193 bond of beta-casein is the bond in the casein system most susceptible to chymosin action. Cleavage of this bond produces a large polypeptide, beta-CN (f1-192) and a short peptide (f193-209) which is hydrophobic and extremely bitter.

The rate of cleavage of the Leu192-Tyr193 bond is very dependent on the ionic strength of the cheese aqueous phase. Increasing ionic strength by increasing NaCl concentration results in increased hydrophobic interaction between the hydrophobic C-terminal regions of beta-casein thus tending to block the action of chymosin and reducing the production of beta-CN (f193-209) and related peptides and thus bitterness.

Hence (and strangely), the first question to ask if a cheese is bitter, is what is its salt content!

Note: "beta" and "kappa" above are usually written as Greek letters but I cannot seem to use them on this site. The photo above shows the amino acid sequence of the C-terminal region of bovine beta-casein with arrows indicating some cleavage sites of chymosin on this protein.