Characteristics of good rennet substitutes

Throughout the twentieth century, cheese production increased steadily but the supply of calf vells (stomachs) did not increase to meet demand for traditional calf rennet. Hence, a search was commenced for rennet substitutes.

The enzymatic coagulation of milk involves destabilisation of the casein micelles by limited proteolysis by enzymes in preparations called rennets. It is not at all unusual to find a proteinase that is able to coagulate milk; indeed, in my experience most proteinases are able to coagulate milk. However, most are most unsuitable for cheesemaking.

The characteristics of a good rennet substitute include:

• Proper specificity. The enzyme must cleave kappa-casein at or near its Phe105-Met106 bond.
• Good activity in milk. The enzyme must be active under the conditions of milk (e.g., have good activity at about pH 6.7).
• Easily denatured in whey. Whey is an increasingly important by-product of cheesemaking (indeed, it is sometimes quipped that "cheese is the by-product of whey manufacture!") It would be highly undesirable to have active coagulant in whey products as the enzyme would be concentrated during whey processing and might cause problems when the whey products are used as ingredients (e.g., in infant formulae).

But most of all, the key criterion of a good rennet substitute is:

• High milk clotting to general proteolysis ratio. In effect, this means that the enzyme must be very specific for the Phe-Met bond of kappa-casein but relatively inactive on peptide bonds elsewhere in the casein system. High proteolytic activity during rennet coagulation will lead to reduction in cheese yield as peptides produced by proteolysis would be lost in the whey rather than being incorporated into the curd. Also, excessive proteolysis by residual coagulant in the cheese can lead to bitterness and perhaps other flavour defects during ripening. In practice, this is the most important criterion for a good rennet substitute and the one least likely to be fulfilled by most proteinases.

I will discuss rennet substitutes in a later post, but briefly, the only enzymes that meet the above criteria to a lesser or greater extent are pepsins (now used rarely), some naturally occurring fungal aspartyl proteinases and, in particular, fermentation-produced chymosins (in which the gene for chymosin is cloned and expressed in a host organism).

Buenos Dias from Buenos Aires

The IV Congreso Internacional de Marketing y Tecnologia de Quesos was held from June 25 to 27 in the wonderful, exciting and vibrant city of Buenos Aires (and amongst my favourite places to visit in the world). The meeting was sponsored principally by Chr Hansen and Fepale (Federacion Panamericana de Lecheria) and included some very useful presentations on cheese science, technology and marketing. Inter alia, Hansen's Chy-Max M, a most interesting coagulant on which we did some work recently, was introduced to the South American market.

It was nice to meet again so many cheese people from Argentina and elsewhere in Latin America. Historically, the Argentine cheese industry was influenced heavily by dairy immigrants from Italy and hence many cheeses of Argentina show a strong family resemblance to Italian varieties. For example, Reggianito Argentino (below) is a tasty variant of Parmesan.

Apart from work, I had a chance to tour Buenos Aires and, at a reception at Chr Hansen's facility, saw the tango danced in its native habitat! Uncle Chris's hospitality was exemplary.

Rennet coagulation of milk

Caseins (~80% of milk protein) occur in milk in the form of large, multi-molecular aggregates called micelles. Casein micelles are approximately spherical aggregates of the 4 types of casein, alpha s1-, alpha s2-, beta- and kappa-casein, together with inorganic ions collectively referred to as colloidal calcium phosphate. There is an uneven distribution of the different caseins throughout the micelle. In particular, kappa-casein is located principally on the surface of the micelle. kappa-Casein stabilizes the micelles and prevents them from aggregating together in the presence of Ca2+. Were it not for kappa-casein, the other caseins would aggregate together as they are highly phosphorylated.

kappa-Casein is divided into 2 parts. Residues 1-105 (approx. two-thirds of the molecule) are hydrophobic and associate with the other caseins. The C-terminal region of kappa-casein (residues 106-169, approximately one-third of the molecule) are hydrophilic (usually containing complex sugar groups esterified to Thr residues) and protrude into the environment, stabilizing the micelle due to steric reasons and the reduction of its zeta-potential.

Enzymatic coagulation of milk involves modification of the casein micelles via limited proteolysis of kappa-casein by proteinase preparations ("rennets") followed by Ca2+-induced aggregation of the rennet-altered micelles.

kappa-Casein is the only casein hydrolyzed during rennet coagulation. kappa-Casein is hydrolyzed at its Phe105-Met106 bond to produce para-kappa-casein (kappa-casein fragment 1-105, kappa-CN f1-105) and macropeptides (also called glycomacropeptides or caseinomacropeptides; kappa-CN f106-169). Macropeptides diffuse into the aqueous phase; para-kappa-casein remains attached to the micelle core. Macropeptides (~30% kappa-casein or 4-5% total casein) are lost. This is an unavoidable loss and a consequence of rennet coagulations but it does have consequences for cheese yield. Proteolysis of kappa-casein by the proteinase(s) in rennet preparations is referred to as the FIRST STAGE OF RENNET ACTION.

Removal of the macropeptides from micelles reduces zeta (surface) potential of the micelles from -20 to about -10 mV and also removes the steric stabilizing layer. When about 85% of total kappa-casein is hydrolyzed, colloidal stability of the micelles is reduced so much that they coagulate at temperatures above about 20oC in the presence of Ca2+. This event is called the SECOND STAGE OF RENNET ACTION.

The Phe105-Met106 bond of kappa-casein is many times more sensitive to rennet action than any other bond in the caseins. Actually, Phe-Met residues aren't necessary; you can replace either amino acid residue in kappa-casein without changing the sensitivity of the bond to rennet action very much. In fact, human, porice and rodent kappa-caseins have Ile or Leu at position 106. Interestingly, the proteinase of C. parasitica does not cleave at Phe105-Met106 but rather Ser104-Phe105 (i.e., the bond next to the Phe-Met bond). Much of the work done in this area has involved synthesizing short peptides with the same amino acid sequence as this region of kappa-casein. The smallest peptide hydrolyzed by rennet is kappa-casein fragment 104-108 (Ser-Phe-Met-Ala-Ile); extending this peptide out towards the N or C-termini of kappa-casein increases its susceptibility to rennet action. Peptide kappa-casin fragment 98-111 is hydrolyzed as well as intact kappa-casein. Certain amino acid residues appear important (e.g., Ser104, Leu103, Ala107, Ile108). The conformation (shape) of kappa-casein renders the Phe-Met bond very susceptible to rennet action.

See also

Fox, P.F. and P.L.H. McSweeney (1998). Dairy Chemistry and Biochemistry. Blackie Academic and Professional Publishers, London, 478 pp. (Reprinted with corrections, 2003.)

Principal steps in cheese manufacture

While some cheeses (e.g., Cottage cheese) are formed from a casein gel formed at the isoelectric point of the caseins, most cheeses are formed from a rennet gel. An important step in cheesemaking is the removal of much of the water from the gel (fat and protein are concentrated approximately 10 fold in many varieties). Dehydration results from a process known as syneresis, in which the rennet (or acid) gel is cut and the gel pieces contract expressing the liquid trapped within (the whey); the solid material is called curds. The process of syneresis is assisted by increasing the temperature of the curds-whey mixture (“cooking”). Cheese curds are salted by brining or by the application of dry salt. Fresh cheese curd has a bland flavour and rubbery texture and it is during the ripening phase (6 months to 2 years for Cheddar) when flavour develops. The principal steps in the manufacture of a rennet-coagulated cheese are shown above.