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Enzyme-Modified Cheese Technology
Enzyme-Modified Cheese Technology G E R A R D J. M O S K O W I T Z and S U E L L E N S. N O E L C K
Dairyland Food Laboratories, Inc. 620 Progress Avenue Waukesha, WI 53186 ABSTRACT
Enzyme-modified cheese is derived from cheese by enzymatic means. Enzymes may be added during the manufacture of cheese or after aging. An incubation period under controlled conditions is required for proper flavor development. The mechanism of flavor development in enzyme-modified cheese m a y be related to the curing of cheese. Although m a n y of the mechanisms for flavor development in cheese are not well understood, carbohydrates, proteins, and fat undergo enzymatic degradation during cheese aging, and these reactions are important in the development of flavor in cheese and enzyme-modified cheese. In some instances, the flavor profile or intensity is proportional to the degree of lipolysis and release of low molecular weight free fatty acids as with Romano or Provolone cheese. In other cases, a similar free fatty acid profile enhances both Cheddar flavor and Swiss cheese flavor but is not characteristic for either. Enzyme-modified cheeses are generally added to foods at levels of .1 to 2.0%, although they can be used at 5% of the formulation to add dairy or cheesy notes to foods and to reduce the requirement for aged cheese in food formulations. INTRODUCTION
Cheese that has been treated enzymatically to enhance the flavor or a significant portion of the flavor profile can be called enzymemodified cheese. Enzyme may be added during the manufacture of the cheese or after the cheese curd has been pressed or even after a period .~f aging. Enzyme-modified cheese may have a texture similar to or slightly modified
Received August 11, 1986. Accepted December 4, 1986.
1987 J Dairy Sci 7 0 : 1 7 6 1 - 1 7 6 9
from the texture of the variety of cheese it represents or it may be in a paste form. Flavor development in cheese is a subject of intense study. The complex enzymatic and nonenzymatic reactions that occur during curing are not well understood. Several hundred compounds have been identified as important components of cheese flavor y e t few of them characterize a particular cheese flavor. Enzymes may be added at various stages during manufacture of cheese or to melted cheese or cheese curds. The nature of the product is affected by the choice of enzymes, conditions of incubation, and the stage at which the enzyme is added. A standard of identity cheese having a typical flavor profile can be produced by the addition of enzymes to milk or fresh curd. The production of this cheese can be accelerated by subjecting it to a controlled aging process. Alternatively, the same process may be used to produce a highly flavored cheese by alteration of the conditions of manufacture. Generally, this cheese also has a modified texture and body. Several months of aging are usually required for flavor development in each case. High flavor intensity, enzyme-modified cheese can also be produced in a matter of days by careful addition of enzymes to the curd or by addition to melted cheese followed by a controlled incubation process. A wide variety of flavors can be produced using enzyme technology and, in fact, a number of different enzyme-modified cheese flavors are commercially available including mild, medium, and sharp Cheddar, as well as Colby, Swiss, Provolone, Romano, Mozzarella, Parmesan, and Brick. Flavor profiles can be tailored to fit an individual customer's needs. Flavor Compounds of Cheese
The chemistry of cheese flavor is very complex. Over 180 compounds have been identified as components of Cheddar cheese
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MOSKOWITZ AND NOELCK
flavor and over 125 compounds as components o f Swiss cheese flavor. Many of these compounds are in both types and as well as in other cheese. It is, therefore, very difficult to characterize chemically cheese flavors. However, there are certain compounds that can be considered significant contributors to a particular cheese-type flavor. Many of the compounds produced during ripening are in concentrations and relative proportions typical for the variety of cheese. A number of these reactions can be accelerated through the use of enzymes. Enzyme-modified cheese will contain almost all of these compounds; however, their relative concentrations may vary, depending on the conditions used to manufacture the product. Therefore, it is important to understand these concepts in cheese flavor development in order to understand the chemistry of flavor and flavor development in enzyme-modified cheese. Some of the important compounds identified in Cheddar cheese flavor are free fatty acids, methanethioI, dimethyl sulfide, diacetyl, butanone, 2-pentanone, lactic acid, acetic acid, and products of protein hydrolysis. Manning (11) followed the development of a number of volatile components by headspace analysis. In general, low molecular weight sulfur compounds increased as the Cheddar cheese aged, although the concentrations tended to level off or decrease as the cheese approached 12 mo of age. It was suggested that these low molecular weight sulfur compounds served as substrates for further nonenzymatic chemical reactions. Methanol, acetone, and 2-pentanone concentrations increased with cheese age and may be
TABLE 1. Important compounds identified as components of swiss cheese flavor. Low molecular weight, free fatty acids, principally: Acetic acid Proprionic acid Butyric acid Diacetyl Proline Amino acids and small peptides Dimethylsulfide lsobutyric acid Isovaleric acid Short-chain thioesters
considered important indicators of flavor development. Proline and proprionic acid are important compounds in Swiss cheese flavor (Table 1). Mitchell (12) indicated that acetic acid, proprionic acid, and proline concentrations increased during aging and he attempted to correlate the relative levels o f each to Swiss flavor. Diacetyl production decreased rapidly within the first few days and remained low throughout ripening. A number of compounds are important contributors to Blue cheese flavor (acetic acid, butanoic acid, acetone, methyl ketones, 2pentanol, methyl hexanoate, ethyl butanoate, 2-nonanol, and free fatty acids). The characteristic flavor is attributed to the production of methyl ketones and corresponding secondary alcohols. These compounds are derived from intermediates of the fatty acid /~-oxidation pathway as outlined in Figure 1. Lipase enzymes are secreted by the developing mycelia
Fatty Acid
Triglyceride Lipase
1
) B-Ketoacyl CoA B-Oxidation
Thiolase
Free ~-Ketoacid
Secondary Alcohol <
Methyl Ketone
Decarboxylase
Reduction
Figure 1. Production of methyl ketones and secondary alcohols ~om free fatty acids by PenicHlium roquefortii. CoA = Coenzyme A.
Journal of Dairy Science Vol. 70, No. 8, 1987
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
Milk protein
peptides Rennet microbial proteases
Flavor ~_ Compounds"
i
enzymatic-and nonenzymatic systems
.% bacterial " peptidases
flavor compound intermediates
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small peptides Amino acids
Flavor precursors by decarboxylation transamination deamination Amino Acid side chain catabolism demethiolation
Figure 2. Lactic cultures and their poslutated role in flavor development in cheese.
and hydrolyze the triglycerides of milk fat. The resulting fatty acids are oxidized to /3-keto acids by the 3-oxidative enzymes of fatty acid degradation. Methyl ketones are produced by decarboxylation of the 3-keto acid. Secondary alcohols are produced from the fl-keto acid by reduction of the carbonyl group. The Cs to C11 methyl ketones and secondary alcohols are very important components of Blue cheese flavor. Starter Enzymes in Cheese Flavor Development
Starter bacteria are required for flavor development in cheese (14). During ripening, starter bacteria die, and as they Iyse, they release their enzymes into the curd. Cheddar flavor may develop as a reult of these enzymatic reactions occurring in the body of the cheese, or in the remaining intact cells, or both. A generalized mechanism of flavor development is outlined by Figure 2. Milk proteins are degraded in a series of reactions by proteolytic enzymes to produce flavor precursors, which are converted to flavor compounds by enzymatic mechanisms. As an example of enzymatic conversion, lactic streptococci convert citrate to diacetyl by a series o.f metabolic reactions involving decarboxylation, high energy intermediates, and reduction (Figure 3). Recent evidence now suggests that Cheddar cheese flavor is also a direct result of nonenzymatic reactions occurring in the body of the cheese. It is thought that the released starter enzymes create the correct environmental conditions for these as yet unknown
nonenzymatic reactions to occur (15). A reducing low redox potential environment is required for correct Cheddar flavor development. It is postulated that starter enzymes create this environment through a series of metabolic steps, which reduces oxygen in the cheese body, and through the production of reduced sulfur compounds. In model Cheddar systems, reduced glutathione is important for flavor development. Harper and Kristoffersen (6) found that glutathione addition enhanced production of cheese flavor in a cheese slurry prepared from fresh curd. They attributed this effect to the ability of glutathione to dissociate proteins, which resulted in an increased rate of proteolysis. In addition, it was postulated that reduced glutathione protected esterases from degradation and maintained flavor compounds in a reduced state. Role of Enzymes in Cheese Flavor Development
Lipolysis is an important reaction in flavor development of cheese. Pregastric esterases derived from the glottal region of calf, kid, and lamb (4, 5) are added to milk during the manufacture of Italian cheeses such as Romano, Provolone, and Parmesan to produce free fatty acids. These free fat W acids are major contributors to the characteristic flavor of the cheese (1). The fatty acid profile of samples of these cheese are outlined in Table 2 (19). Woo and Lindsay (19) found that the fatty acid composition was related to flavor quality and Journal of Dairy Science Vol. 70, No. 8, 1987
o~
o~
< o
TABLE 2. Free fatty acid (FFA) composition and flavors of retail samples of Italian cheese varieties (19). Concentration of FFA (ppm)
Z
o
,,o 00
Cheese variety
Sample code
C4:0
C6:0
C8:0
C,o:o
C12:0
C14:0
C1~:0
C18 Congeners
Provolone
C
782
308
81
172
122
120
199
334
Parmesan
B
502
174
98
223
163
368
621
662
Romano
A
1756
843
328
942
428
448
785
1224
Mozzarella t
A
48
0
6
10
26
72
147
156
* Mozzarella sample was made from whole and part skim milk, respectively.
Flavor Very balanced FFA flavor Strong flavor, lacks balance Full blended flavor, smooth Bland, flat, milky
O © ~ N > X Z © Oq t-
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
Citrate_~
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. Pyruvate
Oxaloacetate
i Ace tate
TPP ~CO
2
Acetaldehyde-TPP yI-CoA CoASH TPP ,3 B u t y l e n e glycol ~L
~
~, Acetoin (
NADH
NAD
o
Diacetyl
NADH~ NAD
Figure 3. Biosynthetic pathway of the lactic streptococci for the production of diacetyl from citrate (16). CoA = Coenzyme A, TPP = thiamine pyrophosphate.
intensity but did not correlate with the age of the cheese. Free fatty acids have also been described as important components of Cheddar flavor. Lipolysis is probably initiated b y milk lipases or lipases from adventitious bacteria. Starter lipases do not hydrolyze triglycerides. Starter lipases can attack the monoglycerides and diglycerides produced by milk or microbial lipases and probably contribute to the development of a typical Cheddar flavor (17). Propionate and acetate are important components of Swiss cheese flavor. Higher fatty acids (C4 to C l s ) are also present in substantial quantities and contribute to the flavor of the cheese (2). Proteolysis is an important process in cheese flavor development. The general role o f starter proteases has been described. Proteases secreted by the cell are found in the medium, on the cell wall, and in the space between the cell wall and cell membrane. Peptidases are also present in the membrane-cell wall area and in the cytoplasm of the starter bacteria (3, 18). These enzymes, in conjunction with rennet and milk proteases, produce a parital hydrolysis of casein, which results in the flavor and texture modifications typical of aged cheese (Figure 4).
Flavor Development in Enzyme-Modified Cheese
A number of reactions that occur during the curing of cheese are important in the development of the flavor of enzyme-modified cheese. Kosikowski and lwasaki (7) evaluated a number o f commercially available enzymes in accelerated Cheddar ,cheese flavor development. They found that different enzyme systems produced different effects when added to Cheddar curds (Table 3). Animal pregastric esterases and peptidases contributed to the flavor of Cheddar cheese while other enzymes produced flavor defects. Law (8) described the acceleration of Cheddar cheese flavor development using bacterial neutral protease. Further refinements included incorporation of a peptidase fraction isolated from Streptococcus lactis, which did not increase the overall breakdown of proteins but did contribute to the development of low molecular weight nitrogenous compounds (9). However, lipase addition to Cheddar cheese during manufacture did not improve the flavor nor did it accelerate the development of flavor either in the presence or absence of added protease (lo). In many instances, lipolysis is important in Jo~arnal of Dairy Science Vol. 70, No. 8, 1987
1766
MOSKOWITZ AND NOELCK CELL WALL
CHEESE
CELL
CYTOPLASM
MEMBRANE
•
I I
°
°
• °
•
•
•
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,~
I
°
it ¢t) klJ
•
•
;I
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°,
•
• PEPT I DASES. PROTEASE
r~
J
Oill a.
oP
,
l
'
.
•
I
HIGH MOLECULAR WEIGHT PEPTIDES
PROTEIN
4
) LOW MOLECULAR WEIGHT PEPTIDES
)
| t
!
•
I o
i
!
A M I N O ACIDS FLAVOR COMPOUNDS
Figure 4. Proteolytic degradation of proteins by starter microorganisms in cheese.
the prodution of enzyme-modified cheese• Studies have indicated that lipolysis contributes significantly to the flavor perception of enzymemodified cheese• The flavor profile of Romano, Parmesan, and Provolone enzyme-modified cheeses are significantly enhanced by the production of the low molecular weight free fatty acids• Lipolysis is also important for the flavor of enzyme-modified Swiss and Cheddar cheese; however, the effect appears to be less specific than for Italian varieties. Table 4 demonstrates the significant increase in fatty acids produced by lipolytic modification• In these examples, butyric acid levels have been elevated 10- to 100-fold, and palmitic acid has been elevated 8- to 50-fold. The degree of
lipolysis is determined by the intensity of flavor desired and the application of the product. As indicated in Table 4, the fatty acid levels of both Swiss and Cheddar have been increased significantly and the overall flavor has been increase more than 10x. However, the relative ratios of free fatty acids for both Cheddar and Swiss enzyme modified cheeses are similar yet one tastes like Cheddar flavor and the other like Swiss flavor (Table 5). It would appear, therefore, that the relative proportions of free fatty acids are critical for Romano, Parmesan, and Provolone enzymemodified cheese flavor but not for Cheddar and Swiss enzyme-modified cheese. Thus, free fatty
TABLE 3. Enzyme-accelerated ripening of Cheddar cheese (7). Enzyme system
Cheese flavor
Decarboxylases Microbial acid proteases Neutral protease Animal esterases Lipases Pep tid ase s
Chemical Bitterness Increased Cheddar flavor Nonspecific flavor and rancidity Cheddar flavor
Journal of Dairy Science Vol. 70, No. 8, 1987
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TABLE 4. Free fatty acid composition of young Swiss and Cheddar cheese and enzyme-modified cheese (EMC).
Fatty acid
60-d Swiss
EMC Swiss
60 to 90-d Cheddar
EMC Cheddar
(mol/lO0 g dry weight) Acetic Proprionic Butyric Caproic Caprylic Cupric Laurie Myristic Palmitic Stearic Oleic Linoleic Linolenic
75.0 105.4 6.6 1.7 . .. 2.6 2.0 6.1 15.2 .5 11.7 2.1 1.4
76.7 89.2 69.0 12.1 11.1 21.8 18.8 44.7 124.2 48.4 101.4 5.4 7.2
acids, a l t h o u g h n o t t h e p r i n c i p a l c h a r a c t e r i s t i c flavor c o m p o n e n t o f C h e d d a r or Swiss, nevert h e l e s s play a m a j o r role in flavor p e r c e p t i o n a n d i n t e n s i t y . This role m a y b e n o n s p e c i f i c . Free f a t t y acids or t h e m o n o g l y c e r i d e s a n d diglycerides m a y assist in t h e e m u l s i f i c a t i o n o f s o m e o f t h e flavor c o m p o n e n t s , or t h e f a t t y acids t h e m s e l v e s m a y serve as s u b s t r a t e s f o r f u r t h e r reactions. A f t e r l i m i t e d h y d r o l y s i s , t h e resulting fat phase may be more capable of dissolving flavor c o m p o u n d s , t h e r e b y i n c r e a s i n g t h e p e r c e p t i o n o f flavor. T h e role o f p r o t e o l y s i s in e n z y m e - m o d i f i e d cheese m a n u f a c t u r e is u n c l e a r . T h e c o n d i t i o n s
1.3 .7 ... .5 .7 2.3 5.7 1.6 5.0 .8 .3
63.3 -.. 169.1 34.0 21.1 52.1 51,6 137.0 285.5 85.7 210.3 17.3 20.1
o f processing d i f f e r f r o m t h o s e used to acc e l e r a t e flavor in cheese aging p r o g r a m s , a n d t h e results can n o t b e d i r e c t l y c o r r e l a t e d . A s i n d i c a t e d in T a b l e 6. p r o t e o l y s i s c o n t r i b u t e s to t h e flavor d e v e l o p m e n t in C h e d d a r a n d Swiss e n z y m e - m o d i f i e d cheese b u t n o t in Blue. T h e p r o d u c t i o n o f e n z y m e - m o d i f i e d cheese remains, t h e r e f o r e , as m u c h o f an art as a science.
Manufactu re of Enzyme-Modified Cheese
As i n d i c a t e d previously, e n z y m e - m o d i f i e d cheese m a y b e p r e p a r e d f r o m cheese t h a t has
TABLE 5. Fatty acid ratios of enzyme-modified cheese (EMC). Butyric acid relative ratio Fatty acid
EMC Cheddar
EMC Swiss
Acetic Butyric Caproic Caprylic Cupric Lauric Myristic Palmitic Stearic Oleic Linoleic Linolenic
.37 1.00 .20 .12 .31 .30 .81 1.69 .51 1.24 .10 .12
1.11 1.0 .17 .16 .31 .27 .65 1.79 .70 1.47 .08 .10
Journal of Dairy Science Vol. 70, No. 8, 1987
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MOSKOWITZ AND NOELCK
TABLE 6. Nonprotein nitrogen content of enzyme-modified cheese.
Cheese type
Nonprotein nitrogen enzyme-modified cheese
Cheese
(mg/g) Cheddar Swiss Blue
6.39 6.66 18.1
16.7 17.6 20.7
TABLE 7. Major Steps in the Manufacture of Lipolyzed Products (13). 1. Prepare the substrate. 2. Mix the enzyme into an aqueous solution and standardize its activity. 3. Add enzyme to substrate. 4. Homogenize the mixture to promote the formation of an emulsion, which is needed to increase enzyme activity. 5. Incubate the emulsified substrate to develop desired flavor in endproducts. 6. Inactivate the enzyme. 7. Standardize the final product. 8. Package.
been enzymatically m o d i f i e d during the manufacture of the curd. Alternately, the cheese itself m a y be modified. A general outline prepared by Nelson (13) describes the m a j o r steps involved. In general, the e n z y m e and substrate are treated to enhance the interaction. A f t e r incubation under controlled conditions, t h e p r o d u c t is treated to inactivate the enzymes, standardized for flavor, and packaged (Table 7). Uses for EnzymeModified Cheese
C o m m e r c i a l e n z y m e - m o d i f i e d cheese have a v e r y intense cheese flavor. In general, t h e y are 5 to 25× the flavor of the typical cheese t h e y represent, although claims of up to 50× the flavor level of the cheese t y p e have been made. The flavor profile is n o t that of a table cheese b u t rather represents an exaggeration of some o f the flavor profiles of the cheese. F o r this reason, e n z y m e - m o d i f i e d cheese are generally used in f o r m u l a t e d or processed foods or as w h o l e or partial substitutes for aged cheese. Typical uses are outlined in the examples that follow. Use ranges f r o m less than 1% o f the formulation to a few percent. F o r a cheese ravioli filling Journal of Dairy Science Vol. 70, No. 8, 1987
(Table 8), 2% o f R o m a n o or Parmesan cheese paste flavor (CPF) is r e c o m m e n d e d ; for a Danish cheese filling (Table 9), .5% of cream cheese CPF is r e c o m m e n d e d . E n z y m e - m o d i f i e d cheeses are particularly useful in cheese p o w d e r formulations or in salad dressings. E n z y m e - m o d i f i e d cheese provides the f o o d m a n u f a c t u r e r with a strong cheese n o t e that is
TABLE 8. Application of enzyme-modified cheese in an Italian cheese spread. Ingredients DFL CPF ® 74004 Romano DFL CPF ® 75005 Parmesan Cheddar cheese, 3 mo Water (divided) Monterey Jack cheese Heavy cream Sweet whey Provolone cheese Disodium phosphate Salt Vegetable gum Mustard flour Total
% Weight .60 .60 30.35 25.40 13.00 12.00 8.25 7.65 1.00 .75 .30 .10 100.00
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
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TABLE 9. Application of enzyme-modified cheese in a Cheddar cheese sauce.
TABLE 10. Application of enzyme-modified cheese in a nacho seasoning.
Ingredients
% Weight
Ingredients
% Weight
DFL CPF ® 71146 Cheddar Water (divided) Cheddar, 3 mo Partially hydrogenated soybean oil Sweet whey Cheddar, 9 mo Modified starch Salt (divided) Monoglycerides and diglycerides Disodium phosphate Autolyzed yeast extract Monosodium glutamate Mustard flour Annatto, double strength
1.10 43.34 32.10 9.00 6.00 4.00 2.40 .70 .60 .40 .10 .10 .10 .06
DFL CPF ® 71003 Cheddar DFL CPF ® 75005 Parmesan DFL CPF ® 74004 Romano Cornstarch Salt Sweet whey All purpose flour Buttermilk powder Tricalcium phosphate Monosodium glutamate Disodium phosphate Lactic acid, 88% Autolyzed yeast extract Color Ground oregano Ground cumin
5.00 2.50 1.25 20.00 20.00 16.60 15.50 10.00 3.00 2.00 1.50 1.25 .50 .50 .35 .05
Total
100.00
Total
cost effective, n u t r i t i o u s , and a natural flavor. T h e range o f use o f t h e s e p r o d u c t s is l i m i t e d o n l y b y t h e i m a g i n a t i o n o f t h e f o o d scientist. As m o r e is learned a b o u t cheese flavor, n e w a n d i m p r o v e d f o r m s o f e n z y m e - m o d i f i e d cheese will b e d e v e l o p e d , o f f e r i n g t h e f o o d i n d u s t r y a w i d e variety o f flavor p r o f i l e s and t y p e s and provide t h e m a n u f a c t u r e r w i t h an i m p r o v e d ability to c u s t o m design t h e s e b i o t e c h n o logically derived ingredients.
10
11
12
REFERENCES
1 Arnold, R. G., K. M. Shahani, and B. K. Dwivedi. 1975. Application of lipolytic enzymes to flavor development in dairy products. J. Dairy Sci. 58:1127. 2 Biede, S. L., and E. G. Hammond. 1979. Swiss cheese flavor 11. organoleptic analysis. J. Dairy Sci. 62:238. 3 Exterkate, F. A. 1984. Location of peptidases outside and inside the membrane of Streptococcus cremoris. Appl. Environ. Microbiol. 47:177. 4 Farnham, M. G. 1950. US Pat. 2,531,329. 5 Farnham, M. G. 1957. US Pat. 2,794,743. 6 Harper, W. J., and T. Kristoffersen. 1970. Biochemical aspects of flavor development in Cheddar cheese slurries. J. Agric. Food Chem. 18:563. 7 Kosikowski, F. V., and T. Iwasaki. 1975. Changes in Cheddar cheese by commercial enzyme preparations. J. Dairy Sci. 58:963. 8 Law, B. A., and A. Wigrnore. 1982. Accelerated cheese ripening with food grade proteinases. J. Dairy Res. 49:137. 9 Law, B. A., and A. Wigmore. 1983. Accelerated ripening of Cheddar cheese with a commercial
13
14
15 16
17 18
19
100.00
proteinase and intracellular enzymes from starter Streptococci. J. Dairy Res. 50:519. Law, B. A., and A. Wigmore. 1985. Effect of commercial lipolytic enzymes on flavor development in Cheddar cheese. J. Soc. Dairy Technol. 38:86. Manning, D. J. 1978. Cheddar cheese flavor studies. I. production of volatiles and development of flavour during ripening. J. Dairy Res. 45:479. Mitchell, G. E. 1981. The production of selected compounds in a Swiss type cheese and their contribution to cheese flavour. Aust. J. Dairy Technol. 36:21. Nelson, J. H. 1972. Enzymaticatiy produced flavors for fatty systems. J. Am. Oil Chem. Soc. 49:559. Reiter, B., T. F. Fryer, A. Pickering, H. R. Chapman, R. C. Lawrence, and M. E. Sharpe. 1967. The effect of the microbial flora on the flavour and free fatty acid composition of Cheddar cheese. J. Dairy Res. 34:257. Sharpe, M. E. 1979. Lactic acid bacteria in the dairy industry. J. Soc. Dairy Technol. 32:9. Speckman, R. A., and E. B. Collins. 1968. Diacetyl biosynthesis in Streptococcus diacetylactis and Leuconostoc citrovorum. J. Bacteriol. 95:174. Stadhouders, J., and H. A. Veringa. Fat hydrolysis by lactic acid bacteria in cheese. 1973. Neth. Milk and Dairy J. 27:77. Thomas, T. D., and O. E. Mills. 1981. Proteolytic enzymes of starter bacteria. Neth. Milk and Dairy J. 35:255. Woo, A. H., and R. C. Lindsay. 1984. Concentration of major free fatty acids and flavor development in Italian cheese varieties. J. Dairy Sci. 67:960.
Journal of Dairy Science Vol. 70, No. 8, 1987
Dairyland Food Laboratories, Inc. 620 Progress Avenue Waukesha, WI 53186 ABSTRACT
Enzyme-modified cheese is derived from cheese by enzymatic means. Enzymes may be added during the manufacture of cheese or after aging. An incubation period under controlled conditions is required for proper flavor development. The mechanism of flavor development in enzyme-modified cheese m a y be related to the curing of cheese. Although m a n y of the mechanisms for flavor development in cheese are not well understood, carbohydrates, proteins, and fat undergo enzymatic degradation during cheese aging, and these reactions are important in the development of flavor in cheese and enzyme-modified cheese. In some instances, the flavor profile or intensity is proportional to the degree of lipolysis and release of low molecular weight free fatty acids as with Romano or Provolone cheese. In other cases, a similar free fatty acid profile enhances both Cheddar flavor and Swiss cheese flavor but is not characteristic for either. Enzyme-modified cheeses are generally added to foods at levels of .1 to 2.0%, although they can be used at 5% of the formulation to add dairy or cheesy notes to foods and to reduce the requirement for aged cheese in food formulations. INTRODUCTION
Cheese that has been treated enzymatically to enhance the flavor or a significant portion of the flavor profile can be called enzymemodified cheese. Enzyme may be added during the manufacture of the cheese or after the cheese curd has been pressed or even after a period .~f aging. Enzyme-modified cheese may have a texture similar to or slightly modified
Received August 11, 1986. Accepted December 4, 1986.
1987 J Dairy Sci 7 0 : 1 7 6 1 - 1 7 6 9
from the texture of the variety of cheese it represents or it may be in a paste form. Flavor development in cheese is a subject of intense study. The complex enzymatic and nonenzymatic reactions that occur during curing are not well understood. Several hundred compounds have been identified as important components of cheese flavor y e t few of them characterize a particular cheese flavor. Enzymes may be added at various stages during manufacture of cheese or to melted cheese or cheese curds. The nature of the product is affected by the choice of enzymes, conditions of incubation, and the stage at which the enzyme is added. A standard of identity cheese having a typical flavor profile can be produced by the addition of enzymes to milk or fresh curd. The production of this cheese can be accelerated by subjecting it to a controlled aging process. Alternatively, the same process may be used to produce a highly flavored cheese by alteration of the conditions of manufacture. Generally, this cheese also has a modified texture and body. Several months of aging are usually required for flavor development in each case. High flavor intensity, enzyme-modified cheese can also be produced in a matter of days by careful addition of enzymes to the curd or by addition to melted cheese followed by a controlled incubation process. A wide variety of flavors can be produced using enzyme technology and, in fact, a number of different enzyme-modified cheese flavors are commercially available including mild, medium, and sharp Cheddar, as well as Colby, Swiss, Provolone, Romano, Mozzarella, Parmesan, and Brick. Flavor profiles can be tailored to fit an individual customer's needs. Flavor Compounds of Cheese
The chemistry of cheese flavor is very complex. Over 180 compounds have been identified as components of Cheddar cheese
1761
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MOSKOWITZ AND NOELCK
flavor and over 125 compounds as components o f Swiss cheese flavor. Many of these compounds are in both types and as well as in other cheese. It is, therefore, very difficult to characterize chemically cheese flavors. However, there are certain compounds that can be considered significant contributors to a particular cheese-type flavor. Many of the compounds produced during ripening are in concentrations and relative proportions typical for the variety of cheese. A number of these reactions can be accelerated through the use of enzymes. Enzyme-modified cheese will contain almost all of these compounds; however, their relative concentrations may vary, depending on the conditions used to manufacture the product. Therefore, it is important to understand these concepts in cheese flavor development in order to understand the chemistry of flavor and flavor development in enzyme-modified cheese. Some of the important compounds identified in Cheddar cheese flavor are free fatty acids, methanethioI, dimethyl sulfide, diacetyl, butanone, 2-pentanone, lactic acid, acetic acid, and products of protein hydrolysis. Manning (11) followed the development of a number of volatile components by headspace analysis. In general, low molecular weight sulfur compounds increased as the Cheddar cheese aged, although the concentrations tended to level off or decrease as the cheese approached 12 mo of age. It was suggested that these low molecular weight sulfur compounds served as substrates for further nonenzymatic chemical reactions. Methanol, acetone, and 2-pentanone concentrations increased with cheese age and may be
TABLE 1. Important compounds identified as components of swiss cheese flavor. Low molecular weight, free fatty acids, principally: Acetic acid Proprionic acid Butyric acid Diacetyl Proline Amino acids and small peptides Dimethylsulfide lsobutyric acid Isovaleric acid Short-chain thioesters
considered important indicators of flavor development. Proline and proprionic acid are important compounds in Swiss cheese flavor (Table 1). Mitchell (12) indicated that acetic acid, proprionic acid, and proline concentrations increased during aging and he attempted to correlate the relative levels o f each to Swiss flavor. Diacetyl production decreased rapidly within the first few days and remained low throughout ripening. A number of compounds are important contributors to Blue cheese flavor (acetic acid, butanoic acid, acetone, methyl ketones, 2pentanol, methyl hexanoate, ethyl butanoate, 2-nonanol, and free fatty acids). The characteristic flavor is attributed to the production of methyl ketones and corresponding secondary alcohols. These compounds are derived from intermediates of the fatty acid /~-oxidation pathway as outlined in Figure 1. Lipase enzymes are secreted by the developing mycelia
Fatty Acid
Triglyceride Lipase
1
) B-Ketoacyl CoA B-Oxidation
Thiolase
Free ~-Ketoacid
Secondary Alcohol <
Methyl Ketone
Decarboxylase
Reduction
Figure 1. Production of methyl ketones and secondary alcohols ~om free fatty acids by PenicHlium roquefortii. CoA = Coenzyme A.
Journal of Dairy Science Vol. 70, No. 8, 1987
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
Milk protein
peptides Rennet microbial proteases
Flavor ~_ Compounds"
i
enzymatic-and nonenzymatic systems
.% bacterial " peptidases
flavor compound intermediates
1763
small peptides Amino acids
Flavor precursors by decarboxylation transamination deamination Amino Acid side chain catabolism demethiolation
Figure 2. Lactic cultures and their poslutated role in flavor development in cheese.
and hydrolyze the triglycerides of milk fat. The resulting fatty acids are oxidized to /3-keto acids by the 3-oxidative enzymes of fatty acid degradation. Methyl ketones are produced by decarboxylation of the 3-keto acid. Secondary alcohols are produced from the fl-keto acid by reduction of the carbonyl group. The Cs to C11 methyl ketones and secondary alcohols are very important components of Blue cheese flavor. Starter Enzymes in Cheese Flavor Development
Starter bacteria are required for flavor development in cheese (14). During ripening, starter bacteria die, and as they Iyse, they release their enzymes into the curd. Cheddar flavor may develop as a reult of these enzymatic reactions occurring in the body of the cheese, or in the remaining intact cells, or both. A generalized mechanism of flavor development is outlined by Figure 2. Milk proteins are degraded in a series of reactions by proteolytic enzymes to produce flavor precursors, which are converted to flavor compounds by enzymatic mechanisms. As an example of enzymatic conversion, lactic streptococci convert citrate to diacetyl by a series o.f metabolic reactions involving decarboxylation, high energy intermediates, and reduction (Figure 3). Recent evidence now suggests that Cheddar cheese flavor is also a direct result of nonenzymatic reactions occurring in the body of the cheese. It is thought that the released starter enzymes create the correct environmental conditions for these as yet unknown
nonenzymatic reactions to occur (15). A reducing low redox potential environment is required for correct Cheddar flavor development. It is postulated that starter enzymes create this environment through a series of metabolic steps, which reduces oxygen in the cheese body, and through the production of reduced sulfur compounds. In model Cheddar systems, reduced glutathione is important for flavor development. Harper and Kristoffersen (6) found that glutathione addition enhanced production of cheese flavor in a cheese slurry prepared from fresh curd. They attributed this effect to the ability of glutathione to dissociate proteins, which resulted in an increased rate of proteolysis. In addition, it was postulated that reduced glutathione protected esterases from degradation and maintained flavor compounds in a reduced state. Role of Enzymes in Cheese Flavor Development
Lipolysis is an important reaction in flavor development of cheese. Pregastric esterases derived from the glottal region of calf, kid, and lamb (4, 5) are added to milk during the manufacture of Italian cheeses such as Romano, Provolone, and Parmesan to produce free fatty acids. These free fat W acids are major contributors to the characteristic flavor of the cheese (1). The fatty acid profile of samples of these cheese are outlined in Table 2 (19). Woo and Lindsay (19) found that the fatty acid composition was related to flavor quality and Journal of Dairy Science Vol. 70, No. 8, 1987
o~
o~
< o
TABLE 2. Free fatty acid (FFA) composition and flavors of retail samples of Italian cheese varieties (19). Concentration of FFA (ppm)
Z
o
,,o 00
Cheese variety
Sample code
C4:0
C6:0
C8:0
C,o:o
C12:0
C14:0
C1~:0
C18 Congeners
Provolone
C
782
308
81
172
122
120
199
334
Parmesan
B
502
174
98
223
163
368
621
662
Romano
A
1756
843
328
942
428
448
785
1224
Mozzarella t
A
48
0
6
10
26
72
147
156
* Mozzarella sample was made from whole and part skim milk, respectively.
Flavor Very balanced FFA flavor Strong flavor, lacks balance Full blended flavor, smooth Bland, flat, milky
O © ~ N > X Z © Oq t-
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
Citrate_~
1765
. Pyruvate
Oxaloacetate
i Ace tate
TPP ~CO
2
Acetaldehyde-TPP yI-CoA CoASH TPP ,3 B u t y l e n e glycol ~L
~
~, Acetoin (
NADH
NAD
o
Diacetyl
NADH~ NAD
Figure 3. Biosynthetic pathway of the lactic streptococci for the production of diacetyl from citrate (16). CoA = Coenzyme A, TPP = thiamine pyrophosphate.
intensity but did not correlate with the age of the cheese. Free fatty acids have also been described as important components of Cheddar flavor. Lipolysis is probably initiated b y milk lipases or lipases from adventitious bacteria. Starter lipases do not hydrolyze triglycerides. Starter lipases can attack the monoglycerides and diglycerides produced by milk or microbial lipases and probably contribute to the development of a typical Cheddar flavor (17). Propionate and acetate are important components of Swiss cheese flavor. Higher fatty acids (C4 to C l s ) are also present in substantial quantities and contribute to the flavor of the cheese (2). Proteolysis is an important process in cheese flavor development. The general role o f starter proteases has been described. Proteases secreted by the cell are found in the medium, on the cell wall, and in the space between the cell wall and cell membrane. Peptidases are also present in the membrane-cell wall area and in the cytoplasm of the starter bacteria (3, 18). These enzymes, in conjunction with rennet and milk proteases, produce a parital hydrolysis of casein, which results in the flavor and texture modifications typical of aged cheese (Figure 4).
Flavor Development in Enzyme-Modified Cheese
A number of reactions that occur during the curing of cheese are important in the development of the flavor of enzyme-modified cheese. Kosikowski and lwasaki (7) evaluated a number o f commercially available enzymes in accelerated Cheddar ,cheese flavor development. They found that different enzyme systems produced different effects when added to Cheddar curds (Table 3). Animal pregastric esterases and peptidases contributed to the flavor of Cheddar cheese while other enzymes produced flavor defects. Law (8) described the acceleration of Cheddar cheese flavor development using bacterial neutral protease. Further refinements included incorporation of a peptidase fraction isolated from Streptococcus lactis, which did not increase the overall breakdown of proteins but did contribute to the development of low molecular weight nitrogenous compounds (9). However, lipase addition to Cheddar cheese during manufacture did not improve the flavor nor did it accelerate the development of flavor either in the presence or absence of added protease (lo). In many instances, lipolysis is important in Jo~arnal of Dairy Science Vol. 70, No. 8, 1987
1766
MOSKOWITZ AND NOELCK CELL WALL
CHEESE
CELL
CYTOPLASM
MEMBRANE
•
I I
°
°
• °
•
•
•
'
,~
I
°
it ¢t) klJ
•
•
;I
~
°,
•
• PEPT I DASES. PROTEASE
r~
J
Oill a.
oP
,
l
'
.
•
I
HIGH MOLECULAR WEIGHT PEPTIDES
PROTEIN
4
) LOW MOLECULAR WEIGHT PEPTIDES
)
| t
!
•
I o
i
!
A M I N O ACIDS FLAVOR COMPOUNDS
Figure 4. Proteolytic degradation of proteins by starter microorganisms in cheese.
the prodution of enzyme-modified cheese• Studies have indicated that lipolysis contributes significantly to the flavor perception of enzymemodified cheese• The flavor profile of Romano, Parmesan, and Provolone enzyme-modified cheeses are significantly enhanced by the production of the low molecular weight free fatty acids• Lipolysis is also important for the flavor of enzyme-modified Swiss and Cheddar cheese; however, the effect appears to be less specific than for Italian varieties. Table 4 demonstrates the significant increase in fatty acids produced by lipolytic modification• In these examples, butyric acid levels have been elevated 10- to 100-fold, and palmitic acid has been elevated 8- to 50-fold. The degree of
lipolysis is determined by the intensity of flavor desired and the application of the product. As indicated in Table 4, the fatty acid levels of both Swiss and Cheddar have been increased significantly and the overall flavor has been increase more than 10x. However, the relative ratios of free fatty acids for both Cheddar and Swiss enzyme modified cheeses are similar yet one tastes like Cheddar flavor and the other like Swiss flavor (Table 5). It would appear, therefore, that the relative proportions of free fatty acids are critical for Romano, Parmesan, and Provolone enzymemodified cheese flavor but not for Cheddar and Swiss enzyme-modified cheese. Thus, free fatty
TABLE 3. Enzyme-accelerated ripening of Cheddar cheese (7). Enzyme system
Cheese flavor
Decarboxylases Microbial acid proteases Neutral protease Animal esterases Lipases Pep tid ase s
Chemical Bitterness Increased Cheddar flavor Nonspecific flavor and rancidity Cheddar flavor
Journal of Dairy Science Vol. 70, No. 8, 1987
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
1767
TABLE 4. Free fatty acid composition of young Swiss and Cheddar cheese and enzyme-modified cheese (EMC).
Fatty acid
60-d Swiss
EMC Swiss
60 to 90-d Cheddar
EMC Cheddar
(mol/lO0 g dry weight) Acetic Proprionic Butyric Caproic Caprylic Cupric Laurie Myristic Palmitic Stearic Oleic Linoleic Linolenic
75.0 105.4 6.6 1.7 . .. 2.6 2.0 6.1 15.2 .5 11.7 2.1 1.4
76.7 89.2 69.0 12.1 11.1 21.8 18.8 44.7 124.2 48.4 101.4 5.4 7.2
acids, a l t h o u g h n o t t h e p r i n c i p a l c h a r a c t e r i s t i c flavor c o m p o n e n t o f C h e d d a r or Swiss, nevert h e l e s s play a m a j o r role in flavor p e r c e p t i o n a n d i n t e n s i t y . This role m a y b e n o n s p e c i f i c . Free f a t t y acids or t h e m o n o g l y c e r i d e s a n d diglycerides m a y assist in t h e e m u l s i f i c a t i o n o f s o m e o f t h e flavor c o m p o n e n t s , or t h e f a t t y acids t h e m s e l v e s m a y serve as s u b s t r a t e s f o r f u r t h e r reactions. A f t e r l i m i t e d h y d r o l y s i s , t h e resulting fat phase may be more capable of dissolving flavor c o m p o u n d s , t h e r e b y i n c r e a s i n g t h e p e r c e p t i o n o f flavor. T h e role o f p r o t e o l y s i s in e n z y m e - m o d i f i e d cheese m a n u f a c t u r e is u n c l e a r . T h e c o n d i t i o n s
1.3 .7 ... .5 .7 2.3 5.7 1.6 5.0 .8 .3
63.3 -.. 169.1 34.0 21.1 52.1 51,6 137.0 285.5 85.7 210.3 17.3 20.1
o f processing d i f f e r f r o m t h o s e used to acc e l e r a t e flavor in cheese aging p r o g r a m s , a n d t h e results can n o t b e d i r e c t l y c o r r e l a t e d . A s i n d i c a t e d in T a b l e 6. p r o t e o l y s i s c o n t r i b u t e s to t h e flavor d e v e l o p m e n t in C h e d d a r a n d Swiss e n z y m e - m o d i f i e d cheese b u t n o t in Blue. T h e p r o d u c t i o n o f e n z y m e - m o d i f i e d cheese remains, t h e r e f o r e , as m u c h o f an art as a science.
Manufactu re of Enzyme-Modified Cheese
As i n d i c a t e d previously, e n z y m e - m o d i f i e d cheese m a y b e p r e p a r e d f r o m cheese t h a t has
TABLE 5. Fatty acid ratios of enzyme-modified cheese (EMC). Butyric acid relative ratio Fatty acid
EMC Cheddar
EMC Swiss
Acetic Butyric Caproic Caprylic Cupric Lauric Myristic Palmitic Stearic Oleic Linoleic Linolenic
.37 1.00 .20 .12 .31 .30 .81 1.69 .51 1.24 .10 .12
1.11 1.0 .17 .16 .31 .27 .65 1.79 .70 1.47 .08 .10
Journal of Dairy Science Vol. 70, No. 8, 1987
1768
MOSKOWITZ AND NOELCK
TABLE 6. Nonprotein nitrogen content of enzyme-modified cheese.
Cheese type
Nonprotein nitrogen enzyme-modified cheese
Cheese
(mg/g) Cheddar Swiss Blue
6.39 6.66 18.1
16.7 17.6 20.7
TABLE 7. Major Steps in the Manufacture of Lipolyzed Products (13). 1. Prepare the substrate. 2. Mix the enzyme into an aqueous solution and standardize its activity. 3. Add enzyme to substrate. 4. Homogenize the mixture to promote the formation of an emulsion, which is needed to increase enzyme activity. 5. Incubate the emulsified substrate to develop desired flavor in endproducts. 6. Inactivate the enzyme. 7. Standardize the final product. 8. Package.
been enzymatically m o d i f i e d during the manufacture of the curd. Alternately, the cheese itself m a y be modified. A general outline prepared by Nelson (13) describes the m a j o r steps involved. In general, the e n z y m e and substrate are treated to enhance the interaction. A f t e r incubation under controlled conditions, t h e p r o d u c t is treated to inactivate the enzymes, standardized for flavor, and packaged (Table 7). Uses for EnzymeModified Cheese
C o m m e r c i a l e n z y m e - m o d i f i e d cheese have a v e r y intense cheese flavor. In general, t h e y are 5 to 25× the flavor of the typical cheese t h e y represent, although claims of up to 50× the flavor level of the cheese t y p e have been made. The flavor profile is n o t that of a table cheese b u t rather represents an exaggeration of some o f the flavor profiles of the cheese. F o r this reason, e n z y m e - m o d i f i e d cheese are generally used in f o r m u l a t e d or processed foods or as w h o l e or partial substitutes for aged cheese. Typical uses are outlined in the examples that follow. Use ranges f r o m less than 1% o f the formulation to a few percent. F o r a cheese ravioli filling Journal of Dairy Science Vol. 70, No. 8, 1987
(Table 8), 2% o f R o m a n o or Parmesan cheese paste flavor (CPF) is r e c o m m e n d e d ; for a Danish cheese filling (Table 9), .5% of cream cheese CPF is r e c o m m e n d e d . E n z y m e - m o d i f i e d cheeses are particularly useful in cheese p o w d e r formulations or in salad dressings. E n z y m e - m o d i f i e d cheese provides the f o o d m a n u f a c t u r e r with a strong cheese n o t e that is
TABLE 8. Application of enzyme-modified cheese in an Italian cheese spread. Ingredients DFL CPF ® 74004 Romano DFL CPF ® 75005 Parmesan Cheddar cheese, 3 mo Water (divided) Monterey Jack cheese Heavy cream Sweet whey Provolone cheese Disodium phosphate Salt Vegetable gum Mustard flour Total
% Weight .60 .60 30.35 25.40 13.00 12.00 8.25 7.65 1.00 .75 .30 .10 100.00
SYMPOSIUM: CHEESE RIPENING TECHNOLOGY
1769
TABLE 9. Application of enzyme-modified cheese in a Cheddar cheese sauce.
TABLE 10. Application of enzyme-modified cheese in a nacho seasoning.
Ingredients
% Weight
Ingredients
% Weight
DFL CPF ® 71146 Cheddar Water (divided) Cheddar, 3 mo Partially hydrogenated soybean oil Sweet whey Cheddar, 9 mo Modified starch Salt (divided) Monoglycerides and diglycerides Disodium phosphate Autolyzed yeast extract Monosodium glutamate Mustard flour Annatto, double strength
1.10 43.34 32.10 9.00 6.00 4.00 2.40 .70 .60 .40 .10 .10 .10 .06
DFL CPF ® 71003 Cheddar DFL CPF ® 75005 Parmesan DFL CPF ® 74004 Romano Cornstarch Salt Sweet whey All purpose flour Buttermilk powder Tricalcium phosphate Monosodium glutamate Disodium phosphate Lactic acid, 88% Autolyzed yeast extract Color Ground oregano Ground cumin
5.00 2.50 1.25 20.00 20.00 16.60 15.50 10.00 3.00 2.00 1.50 1.25 .50 .50 .35 .05
Total
100.00
Total
cost effective, n u t r i t i o u s , and a natural flavor. T h e range o f use o f t h e s e p r o d u c t s is l i m i t e d o n l y b y t h e i m a g i n a t i o n o f t h e f o o d scientist. As m o r e is learned a b o u t cheese flavor, n e w a n d i m p r o v e d f o r m s o f e n z y m e - m o d i f i e d cheese will b e d e v e l o p e d , o f f e r i n g t h e f o o d i n d u s t r y a w i d e variety o f flavor p r o f i l e s and t y p e s and provide t h e m a n u f a c t u r e r w i t h an i m p r o v e d ability to c u s t o m design t h e s e b i o t e c h n o logically derived ingredients.
10
11
12
REFERENCES
1 Arnold, R. G., K. M. Shahani, and B. K. Dwivedi. 1975. Application of lipolytic enzymes to flavor development in dairy products. J. Dairy Sci. 58:1127. 2 Biede, S. L., and E. G. Hammond. 1979. Swiss cheese flavor 11. organoleptic analysis. J. Dairy Sci. 62:238. 3 Exterkate, F. A. 1984. Location of peptidases outside and inside the membrane of Streptococcus cremoris. Appl. Environ. Microbiol. 47:177. 4 Farnham, M. G. 1950. US Pat. 2,531,329. 5 Farnham, M. G. 1957. US Pat. 2,794,743. 6 Harper, W. J., and T. Kristoffersen. 1970. Biochemical aspects of flavor development in Cheddar cheese slurries. J. Agric. Food Chem. 18:563. 7 Kosikowski, F. V., and T. Iwasaki. 1975. Changes in Cheddar cheese by commercial enzyme preparations. J. Dairy Sci. 58:963. 8 Law, B. A., and A. Wigrnore. 1982. Accelerated cheese ripening with food grade proteinases. J. Dairy Res. 49:137. 9 Law, B. A., and A. Wigmore. 1983. Accelerated ripening of Cheddar cheese with a commercial
13
14
15 16
17 18
19
100.00
proteinase and intracellular enzymes from starter Streptococci. J. Dairy Res. 50:519. Law, B. A., and A. Wigmore. 1985. Effect of commercial lipolytic enzymes on flavor development in Cheddar cheese. J. Soc. Dairy Technol. 38:86. Manning, D. J. 1978. Cheddar cheese flavor studies. I. production of volatiles and development of flavour during ripening. J. Dairy Res. 45:479. Mitchell, G. E. 1981. The production of selected compounds in a Swiss type cheese and their contribution to cheese flavour. Aust. J. Dairy Technol. 36:21. Nelson, J. H. 1972. Enzymaticatiy produced flavors for fatty systems. J. Am. Oil Chem. Soc. 49:559. Reiter, B., T. F. Fryer, A. Pickering, H. R. Chapman, R. C. Lawrence, and M. E. Sharpe. 1967. The effect of the microbial flora on the flavour and free fatty acid composition of Cheddar cheese. J. Dairy Res. 34:257. Sharpe, M. E. 1979. Lactic acid bacteria in the dairy industry. J. Soc. Dairy Technol. 32:9. Speckman, R. A., and E. B. Collins. 1968. Diacetyl biosynthesis in Streptococcus diacetylactis and Leuconostoc citrovorum. J. Bacteriol. 95:174. Stadhouders, J., and H. A. Veringa. Fat hydrolysis by lactic acid bacteria in cheese. 1973. Neth. Milk and Dairy J. 27:77. Thomas, T. D., and O. E. Mills. 1981. Proteolytic enzymes of starter bacteria. Neth. Milk and Dairy J. 35:255. Woo, A. H., and R. C. Lindsay. 1984. Concentration of major free fatty acids and flavor development in Italian cheese varieties. J. Dairy Sci. 67:960.
Journal of Dairy Science Vol. 70, No. 8, 1987