Food Biochemistry and Food Processing (2 edition)

BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come

228 Part 2: Biotechnology and Enzymology

Chymosin Concentration

Chymosin concentration affects gel firmness (Lomholt and Qvist

1999), and enzyme kinetics have been used to study these effects.

An increase in the chymosin concentration (larger amounts of

rennet added to milk) reduces the total time required for rennet

clotting, measured by the appearance of the first curd particle. As

a result, the secondary phase of rennet action will also proceed

much earlier, with the net result of an increase in the rate of

increase in gel firmness. This property of chymosin is sometimes

used to control the rennet coagulation in concentrated milks,

which have a tendency to form firmer curd because of the higher

protein content. Lowering the amount of rennet reduces the rate

of curd firming. It should also be noted that with an alteration in

the amount of rennet added to milk, there also will be a change

in the amount of residual chymosin in the cheese.

Temperature

The optimal condition for curd formation in milk with chy-

mosin is 40–45◦C, but this temperature is not suitable for cheese

making. Rennet coagulation for cheese making generally oc-

curs at 30–35◦C for proper firmness. At lower temperatures,

rennet clotting rate is significantly reduced, and at refrigeration

temperatures, virtually no curd is obtained.

pH

Chymosin action in milk is optimal at approximately pH 6. This

is obtained with starter bacteria. If the pH is lowered further,

rennet clotting occurs at a faster rate, and curd firmness is re-

duced. Lowering pH also changes the water-holding capacity,

which will have an impact on curd firmness.

Calcium

Calcium has a significant impact on rennet curd, though it does

not have a direct effect on the primary phase of rennet action.

Addition of ionic calcium, as in the form of calcium chloride, for

example, reduces the rate of rennet clot formation time and also

increases rennet curd firmness. Similarly, if the calcium content

of milk is lowered by approximately 30%, coagulation does not

occur. Milks that have a tendency to form weak curd may be

fortified with calcium chloride prior to the addition of rennet.

Milk Processing

Heating milk to temperatures higher than approximately 70◦C

leads to delayed curd formation and weak rennet curd (Vas-

binder et al. 2003). In extreme cases, there may be no curd at

all. These effects are a result of the formation of a complex

involving disulphide linkages between kappa-casein and beta-

lactoglobulin under high heat treatment. Under these conditions,

the 105-106 bond in kappa-casein is inaccessible for chymosin

action. This effect is generally not reversible. Under mild over-

heating conditions, the addition of 0.02% calcium chloride may

help obtain firm rennet curd. Maubois et al. have developed a

process for reversing this effect by the use of ultrafiltration. In-

creasing the protein content by ultrafiltration before or after UHT

treatment restores curd-forming ability (Maubois et al. 1972).

According to Ferron-Baumy et al. (1991), such a phenomenon

results from lowering the zeta potential of casein micelles on

ultrafiltration.

Homogenization has a distinct impact on milk rennet coagu-

lation properties. Homogenized milk produces softer curd, but

when only the cream portion of milk is homogenized, rennet

curd becomes firmer (Nair et al. 2000). This is possibly because

of the reduction of fat globule size due to homogenization and

coating of the fat globule surface with casein. These particles

then act as casein micelles.

Concentrating milk prior to cheese making is now a common

practice. Techniques include evaporative concentration, ultrafil-

tration, and microfiltration. Each of these procedures increases

the casein concentration in milk; thus, the rate of casein aggrega-

tion during the secondary phase increases. Ren-net coagulation

usually occurs at a lower degree of kappa-casein hydrolysis, and

rennet curd is generally firmer. For example, in unconcentrated

milk, approximately 90% kappa-casein must be hydrolyzed by

chymosin before curd formation, but in ultrafiltered milk only

50% must be hydrolyzed. (Dalgleish 1992). Under high concen-

tration conditions, rennet curd is extremely firm, and difficulties

are encountered in cutting curd using traditional equipment. In

cheeses manufactured from highly concentrated milks, rennet

should be properly mixed in the milk mixture to prevent local-

ization of rennet action.

Genetic Variants

Protein polymorphism (genetic variants) of kappa-casein has

been demonstrated to have an effect on rennet coagulation

(Marzialli and Ng-Kwai-Hang 1986). Protein polymorphism

refers to a small variation in the makeup of proteins due to mi-

nor differences in the amino acid sequence. Examples include

kappa-casein AA, AB, or BB, beta-lactoglobulin AA, AB, and

so on. Milk with kappa-casein BB variants forms a firmer rennet

curd because of increased casein content associated with this

variant of kappa-casein. Some breeds of cows, Jerseys in partic-

ular, have larger proportions of this variant. The BB variant of

beta-lactoglobulin is also associated with higher casein content

in milk.

EFFECT OF CHYMOSIN ON

PROTEOLYSIS IN CHEESE

Once rennet curd has been formed and a fresh block of cheese

has been obtained, the ripening process begins. During ripening,

numerous biochemical reactions occur and lead to unique flavor

and texture development. During this process, residual chymosin

that is retained in the cheese makes important contributions

to the ripening process (Kosikowski and Mistry 1997, Sousa

et al. 2001). As the cheese takes form, chymosin hydrolyzes

the paracaseins into peptides optimally at pH 5.6 and creates a

peptide pool for developing flavor complexes (Kosikowski and

Mistry 1997).