BLBS102-c11 BLBS102-Simpson March 21, 2012 13:9 Trim: 276mm X 219mm Printer Name: Yet to Come
11 Chymosin in Cheese Making 225Table 11.1.Examples of Some New Chymosin
ProductsProduct Name Company and TypeAmericanPure Sanofi Bio-Industries
Calf rennet purified via ion exchange
Chy-max Pfizer (Now Chr. Hansen)
Fermentation—usingEscherichia coliK-12
Chymogen Chr. Hansen
Fermentation—usingAspergillus nigervar.
awamori
ChymoStar Rhone-Poulenc (Now Danisco)ˆ
Fermentation—usingAspergillus nigervar.
awamori
Maxiren Gist-Brocades
Fermentation—usingKluyveromyces
marxianusvar.lactis
Novorena Novo Nordisk
(Marzyme GM) Fermentation—usingAspergillus oryzaeaNovoren is not chymosin: it is the coagulating enzyme ofMucor
mieheicloned intoA. oryzae. The other products listed above are all
100% chymosin.rennets, proper cheese flavor might not develop. Most studies
have generally concluded that there are no significant differences
in flavor, texture, composition, and yield compared with calf
rennet controls (Banks 1992, Barbano and Rasmussen 1992,
Biner et al. 1989, Green et al. 1985, Hicks et al. 1988, IDF 1992,
Ortiz de Apodaca et al. 1994).
When these products were first introduced, it was expected
that a majority of cheese makers would convert to these types
of rennets because they are virtually identical to traditional calf
rennet and much cheaper. Except for some areas, this has not gen-
erally happened because of some concerns toward genetically
modified products. Further, some protected cheeses, such as
those under French AOC regulations or Italian DOP regulations,
require the use of only calf rennet. A method has been published
by the International Dairy Federation to detect fermentation-
produced chymosin (Collin et al. 2003). This method uses im-
munochemical techniques (ELISA) to detect such chymosin in
rennet solutions. It cannot be used for cheese.
Thus, traditional calf rennet and rennet substitutes derived
fromMucor Miehei, Cryphonectria, Parasitica, and others are
still used, especially for high cooking temperature cheeses such
as Swiss and Mozzarella. Since the introduction of fermentation
chymosin, the cost of traditional calf rennet has dropped consid-
erably, and they both now cost approximately the same. Fungal
rennets are still available at approximately 65% of the cost of
the recombinant chymosin.
A coagulating enzyme ofMucor mieheicloned intoAs-
pergillus oryzaewas developed by Novo Nordisk of Denmark.
This product hydrolyzes kappa-casein only at the 105-106 bond,
and reduces protein losses to whey.Rennet Production by Separation
of Bovine PepsinTraditional calf rennet contains about 5% pepsin and almost 95%
chymosin. Industrial purification of standard rennet to 100%
chymosin rennet by the removal of pepsin was developed by
Sanofi-Bioingredients Co. The process involves separation of
the pepsin by ion exchange. Chymosin has no charge at pH 4.5,
but bovine pepsin is negatively charged. Traditional commercial
calf rennet is passed through an ion-exchange column contain-
ing positively charged ions. Bovine pepsin, due to its negative
charge, is retained, and chymosin passes through and is col-
lected, resulting in a 100% chymosin product (Pszczola 1989).RENNET SUBSTITUTES
Improved farming practices, including improved genetics, have
led over the years to significant increases in the milk production
capacity of dairy cattle. As a result, while total milk production
in the world has increased, the total cow population has declined.
Hence, there has been a reduction in calf populations and the
availability of traditional calf rennet. Furthermore, the increased
practice of raising calves to an older age for meat production fur-
ther reduced the numbers available for rennet production. It is
for these reasons that a shortage of calf rennet occurred and sub-
stitutes were sought. Various proteolytic enzymes from plant,
microbial, and animal sources were identified and developed
for commercial applications. These enzymes should possess
certain key characteristics to be successful rennet substitutes:
(1) the clotting-to-proteolytic ratio should be similar to that of
chymosin (i.e., the enzyme should have the capacity to clot milk
without being excessively proteolytic); (2) the proteolytic speci-
ficity for beta-casein should be low because otherwise bitterness
will occur in cheese; (3) the substitute product should be free of
contaminating enzymes such as lipases; and (4) cost should be
comparable to or lower than that of traditional rennet.AnimalPepsin derived from swine shows proteolytic activity between
pH 2 and 6.5, but by itself it has difficulty in satisfactorily
coagulating milk at pH 6.6. For this reason, it is used in cheese
making as a 1:1 blend with rennet. Pepsin, used alone as a milk
coagulator, shows high sensitivity to heat and is inclined to create
bitter cheese if the concentrations added are not calculated and
measured exactly (Kosikowski and Mistry 1997).PlantRennet substitutes from plant sources are the least widely used
because of their tendency to be excessively proteolytic and to
cause formation of bitter flavors. Most such enzymes are also
heat stable and require higher setting temperatures in milk. Plant
sources include ficin from the fig tree, papain from the papaya
tree, and bromelin from pineapple. A notable exception to these
problems is the proteolytic enzymes from the flower of thistle
(Cynara Cardunculus), as reported by Vieira de Sa and Barbosa