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Proteolysis and Lipolysis of Goat Milk Cheese

Proteolysis and Lipolysis of Goat Milk Cheese

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J. Dairy Sci. 84(E. Suppl.):E84-E92
 © The American Dairy Science Association, 2001.
Vol. 84, E. Suppl., 2001 E84
Proteolysis and Lipolysisof Goat Milk Cheese
1
 
Young W. Park
Georgia Small Ruminant Research and Extension CenterCollege of Agriculture, Home Economics and Allied ProgramsFort Valley State UniversityFort Valley, GA 31030-4313
ABSTRACT
 Numerous varieties of goat milk cheeses are producedworldwide. Maturation or ripening of goat and other speciesmilk cheeses is governed by interplay of many different fac-tors. Proteolysis and lipolysis are two major biochemical proc-esses in the multifaceted phenomenon of cheese aging, whichinvolves a variety of chemical, physical, and microbiologicalchanges under controlled environmental conditions. Proteoly-sis of cheeses in general is influenced by several factors in-cluding plasmin, chymosin, protease from starter and non-starter bacteria, pH and moisture levels of the curds, storagetemperature and time, salt content, salt-to-moisture ratio, andhumidity. Primary factors affecting lipolysis in cheeses arefatty acid composition, lipolytic enzymes, lipolytic microor-ganisms, moisture, temperature, storage time, oxygen, andsurface area, etc. Several analytical techniques have been usedto measure proteolysis of goat and (or) cow milk cheeses dur-ing ripening, such as solubility of peptides and amino acids invarious solvents or precipitants, liberation of reactive func-tional groups, various forms of chromatography, and differentforms of electrophoresis. Lipolysis of goat milk cheeses has been estimated by acid degree value (ADV), acid value, andfree fatty acid concentration, while lipid oxidation of dairygoat products can be determined by peroxide value, thiobarbu-turic acid value (TBA). Recent reports have shown that goatcheeses had greater rates of protein degradation than cowcounterparts, and that aging time and temperature synergisti-cally elevated most of proteolytic and lipolytic indices in goatcheeses. This paper will further discuss proteolytic andlipolytic characteristics of goat milk cheeses.(
Key words:
proteolysis, lipolysis, goat milk cheese)
Abbreviation key:
 
WSN
= water-soluble nitrogen,
TCA-SN
 = TCA-soluble N,
TBA
= thiobarbituric acid,
ADV
= aciddegree value,
POV
= peroxide value.
INTRODUCTION
Goat milk cheese in the United States has gained popular-ity among ethnic groups, gourmet and health food lovers, andgoat farmers (69), while scientific studies on characteristics of caprine cheeses including proteolysis and lipolysis are stilllimited.As in cow milk cheeses, numerous biochemical and physical changes can occur in goat milk cheeses during distri- bution and storage processes due to ripening and degradationof nutrients in the products. Proteolysis and lipolysis are two primary processes in cheese ripening with a variety of chemi-cal, physical, microbiological, textural, and rheological changesthat take place, usually under controlled environmental condi-tions (9, 34, 46, 58, 59, 72, 80).The rate, extent, and nature of protein and fat degradationof cow milk cheeses during aging have been reviewed exten-sively in several reports (34, 58, 95). Cheese quality is greatlyinfluenced by levels of peptides, amino acids, and free fattyacids resulting from proteolysis and lipolysis (26, 44). Prote-olysis is probably the most important biochemical event, pro-viding a major impact on flavor and texture of most cheesevarieties (34). Hydrolysis of fat catalyzed by enzymes has also been a major dairy industry problem because it can cause ran-cid flavor defect in milk, cheese, and other dairy products (16,85). Flavor deterioration from lipolysis of dairy products cre-ates serious problem in storage stability (3, 23). Althoughmuch data have been documented on the proteolysis andlipolysis of cow milk cheeses, the reports may not be directlyapplicable to goat milk counterparts.The objectives of this paper are 1) to review the major factors involved in proteolysis and lipolysis in cheeses, andthe methods used for analysis of the two aging parameters, and2) to reevaluate the major highlights of recent studies on pro-teolytic and lipolytic characteristics of goat milk cheeses con-ducted mainly in our laboratories.
1. ORIGIN AND VARIETIESOF GOAT MILK CHEESES
 
Goat milk cheese originated in Mesopotamia. The milk was probably made into soft cheese, and then hard, ripenedgoat milk cheeses were developed later in the Mediterranean basin countries, such as Turkey, Greece, Syria, Israel, Iraq,and Iran (51).France produces and exports many types of goat cheeses,including Crottin du Chavignol, Les Pyramides, Sainte Maure,Chabis, and Chabicou (51). The United States has imported1100 metric tones of French goat cheeses in 1998 (8). Other successful goat milk cheese-producing countries includeGreece, Norway, Spain, and Italy. No significant attention wasgiven to the production of commercial goat cheese in theUnited States until 1980.
Received July 26, 2000.Accepted September 28, 2000.E-mail: parky@mail.fvsu.edu.
 
SYMPOSIUM: SMALL RUMINANT RESEARCH: MILK AND DAIRY FOODS
Vol. 84, E. Suppl., 2001 E85
 Numerous varieties of goat milk cheese are producedthroughout the world, depending on diversity in locality, milk composition, and manufacturing techniques used. The USDAAgricultural Handbook No. 54 (82) describes over 400 varie-ties of goat cheeses and lists 800 names of cheeses made fromgoat milk or combinations of goat with other species milk. Thevarietal differences among goat milk cheeses are mainly at-tributed to the nature of physical and chemical changes duringripening (35, 36), which are also influenced by chemicals,cultures or flavor ingredients added to curd during processing(50, 62).The goat cheese industry is classified as a “cottage indus-try’ due to the high moisture content in caprine cheeses, andalmost all French goat cheeses are of the natural drainage type by slow coagulation (60). A recent study of commercial goatcheeses produced in the United States has shown that 20 out of 30 varieties were very high or high moisture cheeses (69).
2. PROTEOLYSIS IN CHEESE
Protein composition in the initial cow or goat milk willhave profound effect on the proteolytic characteristics of rip-ened cheeses. Electrophoretic mobility under standard condi-tions shows that
β
-casein is the major component of caseinfraction of goat milk, whereas
α
sl
-CN is the major componentof cow milk (15, 45). The respective mean
α
sl
-CN and
β
-CNcontents as a percentage of total casein in goat and cow milksare 5.6 and 38; 55 and 36 (76). Goat milk also has higher con-centrations of 
α
s2
- and
κ 
-CN than cow milk. Because of thesecompositional differences, aging and proteolytic properties of the two species milk cheeses would be significantly different.The firmness of cheese curd is strongly correlated with
α
sl
-CN content of milk (45, 76).
β
-Casein is shown to be moreresistant to enzymatic degradation than
α
sl
-CN. The rate of  protein degradation is strongly associated with the accessibil-ity of proteolytic enzymes to the cheese substrates, which isgoverned by the structure and configuration of the cheese pro-tein matrix. Proteolysis in cow cheese has been divided intothree phases, such as before, during, and post manufacture of cheese (34), which may be applicable to goat milk cheeses.
Proteolysis Before Manufacture
Proteolysis can occur in the initial milk before cheesemanufacture. This stage of proteolysis is attributable to twotypes of enzymes, mainly microbial and indigenous milk pro-teinases. Psychrotrophic bacteria are found in raw and pasteur-ized milk. Psychrotrophic bacteria dominate the microflora of milk cooled on-farm before collection, during transportation,and during storage at the dairy plant (34, 61). Psychrotroph populations more than 10
6
cfu/ml are likely to cause reducedrecovery of milk solids as cheese, higher moisture contents, pasty texture, and off-flavors in the cheese (17, 41).Many indigenous proteinases are present in milk. The principal proteinase is plasmin, formally known as alkalinemilk proteinase, which is dissociated with casein micelle (59),and preferentially hydrolyzes
β
-CN to
γ
-CN and proteose pep-tones (34). Most of the proteose-peptone fraction formed by plasmin is lost in acid or rennet whey (57, 59). Plasminogen is present as the precursor of plasmin, which hydrolyzes
α
s2
-casein rapidly, but has little effect on
κ 
-casein that is benefi-cial for milk stability.
α
s1
-Casein may be hydrolyzed slowly to
λ 
-casein (1).
Proteolysis During Manufacture
Milk proteins undergo enzymatic coagulation duringcheese manufacture. Rennet is a mixture of chymosin, pepsin,and other enzymes from calf stomach. Rennet coagulates milk  proteins, which causes proteolysis by the formation of para-
κ 
-CN and NPN.Rennet coagulation is a two-stage process (34). The firstinvolves the enzymatic formation of para-
κ 
-CN and peptides,and the second involves the precipitation of para-
κ 
-CN bycalcium ion at temperature above 20
o
C. Free para-
κ 
-CN is readilyattacked by many enzymes, contributing to a well-ripenedcheese (50). More than one peptide is produced for this prote-olysis, and the substrate is
α
-CN, not
β
-CN (2).
κ 
-Casein isresponsible for micelle stability, but its stabilizing propertiesare lost at renneting (50, 97), and
κ 
-CN is hydrolyzed duringthe primary phase of rennet action (96). For the secondary(nonenzymatic) phase of coagulation, casein micelles begin toaggregate when about 85% of 
κ 
-CN is hydrolyzed (22).
Proteolysis Postmanufacture
Ripening after manufacture causes many changes in the body and flavor of a cheese. Amino acids, fatty acids, methylketones of different carbon length, lactones, and many other organic compounds become free in the cheese and concen-trate, depending on particular cheese types and aging condi-tions (50).Ripening of cheese also causes two distinct phases texturedevelopment. The first phase occurs in the first 7 to 14 d,when the rubbery young cheese curd is rapidly changed to asmoother, more homogeneous texture (59). The casein net-work at this phase is considerably weakened through the hy-drolysis of about 20% of the single bond in the
α
s1
-CN to pro-duce
α
s1
-I by the coagulant (18). For the second phase, a moregradual change occurs in texture because of the degradation of the rest of the
α
s1
-CN during an extended aging period.Proteolysis affects cheese ripening in several ways: 1)changes in texture through degradation of the protein network,increase in pH, and higher water binding by the newly formedamino and carboxyl groups (59), 2) development of flavor components such as amino acids and peptides, some of whichmay cause off-flavors (i.e., bitterness) (63), 3) changes in pH by the formation of NH
3
(49), and 4) higher release of sapidcompounds during mastication (64).
Desirable and UndesirableProteolytic Agents in Cheese Ripening
There are at least five proteolytic agents involved incheese aging. The five proteolytic agents include: a) indige-nous enzymes in raw and (or) pasteurized milks, b) coagulat-ing enzymes, rennet, or its substitute such as chymosin, pepsinor microbial proteinases, c) starter culture bacteria and their enzymes after the cells are lysed, d) enzymes from secondarystarters, such as propionic acid bacteria, and yeasts and molds,and e) nonstarter bacteria opportunistically entered duringcheese making (34). Some organisms cause undesirable de-velopment of open texture and gases in the cheeses.Heterofermentative lactobacilli, yeasts and molds and cit-rate- and spore-forming bacteria are undesirable organismscausing defects of ripened cheeses. They are adventitiouslyintroduced with the starter culture; some survive pasteuriza-tion of milk or gain access to cheese milk or curds from dairy processing equipment and utensils during manufacture (56).
 
PARK
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 
α
s1
-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
β
-CN to
γ
-CN and proteose peptones. Proteinases and peptidasesalso hydrolyze
α
s1
- and
α
s2
-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 
α
s1
-CN was relatively greater at low pHthan that of 
β
-CN.
β
-Casein was more degraded than
α
s1
-CN at pH above 5.6, presumably as a result of elevated plasmin ac-tivity (59).
Salt-to-moisture ratio
.
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
α
s1
- and
β
-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
o
C develops a texture in8 wk equivalent to that developed in 16 wk at 8
o
C (30). At atemperature between 2 and 10
o
C, the texture of Cheddar cheese will not be markedly changed, because
α
s1
-CN is con-sidered a far more important structural element in the cheeseframework than
β
-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.
 Humidity.
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
o
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
o
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).
Chromatographic methods.
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).
 Electrophoresis.
 
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
6
to C
12
) than cow milk (39, 45,71), and the former has smaller fat globule size than the latter 

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