E86 Journal of Dairy Science
MAJOR FACTORS INFLUENCINGTHE RATE OF PROTEOLYSIS
The rate of proteolysis in cheese is highly dependent onthe microbial constituency and the interplay of many other factors (50, 57, 59). Proteolytic activity in cow milk cheesescan be mainly evaluated by the type of coagulant used, exis-tence of residual rennet and native milk proteinases, pH of cheese curd at draining and milling, salt to moisture ratio,temperature of ripening, changes in pH during ripening, redox potential, and mineral (calcium, copper, zinc, and iron) con-tents of the cheese (34, 50, 57, 59). The major factors affecting proteolysis in cheeses can be summarized as follows:
Enzymes (rennet and chymosin) from the starter culture.
The more rennet is retained in the curd, the greater the propor-tion of
-CN is hydrolyzed by the chymosin in rennet (20).
Enzymes from nonstarter and cheese milk (plasmin and other native proteinases).
Cow milk contains many pro-teinases, the principal one being plasmin, which hydrolyzes
-CN and proteose peptones. Proteinases and peptidasesalso hydrolyze
-CN. Most of the proteose peptonefractions are lost in acid or rennet whey (57, 59).
Moisture level of curd.
The rate of ripening is propor-tional to the moisture content of cheese, while the duration of ripening is more or less inversely proportional to the moisturecontent. Small changes in the moisture to casein ratio canmarkedly change the availability of moisture, because a sig-nificant amount of the moisture is bound to the caseins andtheir degradation products (59).
pH of curd.
The shift in pH reflects marked changes inthe nature of newly formed compounds in cheese. The overallextent of proteolysis is increased markedly in simulatedcheese at pH greater than 5.8 (66). The pH is increased at later stages of ripening due to the generation of ammonia (50). Thedegradation rate of
-CN was relatively greater at low pHthan that of
-Casein was more degraded than
-CN at pH above 5.6, presumably as a result of elevated plasmin ac-tivity (59).
Salt content, method of salting,and salt-to-moisture ratio in curd all markedly affect the rateof proteolysis of cheese. An inverse relationship was observed between the degradation rates of both
-CN and theS:M ratio (88).
Aging time and temperature.
The higher the temperature,the greater is the extent of casein hydrolysis and change intexture. Cheddar cheese ripened at 15
C develops a texture in8 wk equivalent to that developed in 16 wk at 8
C (30). At atemperature between 2 and 10
C, the texture of Cheddar cheese will not be markedly changed, because
-CN is con-sidered a far more important structural element in the cheeseframework than
-CN or the other caseins (25). Proteolysis of cheese is inversely correlated with cheese firmness andspringiness, whereby softening of the cheese occurs as the protein matrix is degraded (30). Several recent studies in our laboratory revealed that proteolysis in goat milk cheese is syn-ergistically elevated by the increased temperature and agingtime (47, 72), which will be further discussed in the later partof this paper.
Different types of cheese require different tem- peratures, relative humidities (RH) and times for ripening. For the soft goat milk cheese drying process, an average tem- perature of 15
C combined with 85% RH is usually satisfac-tory in air-conditioned rooms (60). The proper ripening of goat milk cheeses can be achieved at the temperature range of 8 to 12
C, with relatively high humidity ranging from 85 to95% (60).
ANALYTICAL METHODS MEASURINGPROTEOLYSIS IN CHEESE
Several approaches have been adopted for quantitativemeasurement of proteolysis of cheese during ripening. Four major methodologies include: a) solubility of peptides andamino acids in various solvents or precipitants, b) liberation of reactive functional groups, c) several forms of chromatogra- phy, and d) electrophoresis (34, 46).
Solubility of peptides in various solvents or precipitants.
Nitrogen solubility under defined conditions such as fractional precipitation or solubilization is most widely used method of estimating proteolysis (62). The solubility was measured in5% NaCl (37); sodium acetate buffers, pH 4.6 (21, 68); citrate buffers, pH 4.4 (92); 2, 2.5, 4, 10, or 12% TCA (53, 68, 71,76); 50% ethanol (78); 70% ethanol (49); 80% ethanol plus75% acetone (74); 5% phosphotungstic acid (75); and 0.85% picric acid (77).
Liberation of reactive functional groups.
Protein degra-dation may also be measured by monitoring the liberation of amino or carboxyl groups in cheese through reaction withtrinitrobenzene sulphonic acid (81) or ninhydrin (68). Thesereagents can determine a direct consequence of proteolysis byforming amino groups with cheese or its fractions, and are notdependent on an indirect effect of solubility in some particular solvents (34).Another method assaying reactive functional groups ismeasuring certain amino acids using colorimetric procedures.Total tyrosine liberated in aging measured by colorimetricmethod was more sensitive than by the soluble nitrogen method(55). Measurement of the tyrosine content of alcohol-, TCA-,or water-soluble extracts is a well-established method of as-sessing proteolysis. Soluble tyrosine and tryptophan in cheesewere also measured by absorbance at 270 and 290 nm (91).
Various types of chroma-tographic methods have been applied to study fractionation of proteolytic degradative products in cheeses. Those methodsinclude: 1) paper chromatography (52); 2) thin layer chroma-tography (27, 93); 3) ion-exchange chromatography (42); 4)HPLC (6, 10, 13, 14, 43, 92); 5) hydrophobic chromatography(54, 95); 6) gel permeation chromatography (6, 27, 33, 42); 7)cellulose derivatives(19, 55); 8) silica gel chromatography(94).
Sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) has been widely used tostudy casein hydrolysis and the type of proteolysis in cheese, because it has high resolution and can give quantitative results(20, 24, 47, 84, 88), while its quantitative usage was criticizedonce (33). Other types of PAGE were also used to improve theefficiency of the gel procedures, including different buffersand staining methods (24, 60, 84).
3. LIPOLYSIS IN CHEESESAND OTHER DAIRY PRODUCTSLipolysis in Milk
Goat milk contains higher concentrations of short- andmedium-chain fatty acids (C
) than cow milk (39, 45,71), and the former has smaller fat globule size than the latter