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Reduced Fat Cheddar Cheese from Condensed Milk. 3. Accelerated Ripening1
DAIRY FOODS Reduced Fat Cheddar Cheese from Condensed Milk. 3. Accelerated Ripening 1 R. L. BRANDSMA,2 V. V. MISTRY,3 D. L. ANDERSON, and K. A. BALDWIN Minnesota-South Dakota Dairy Foods Research Center Dairy Science Department South Dakota State University Brookings 57007-0647 ABSTRACT
dar cheese, TRT solids.
Reduced fat Cheddar cheeses were manufactured from control uncondensed milk (10.27% total solids) and condensed milks containing 15.40, 18.33, and 22.23% total solids. Three ripening treatments were applied to the cheeses: control, 6 to 7°C; elevated temperature, 11·C; and enzyme added, 6 to 7·C. The cheeses were ripened for up to 12 wk. Soluble nitrogen production was slower in the condensed milk cheeses. Condensed milk cheeses were drier and had crumbly body with increasing milk concentration. Elevated ripening temperatures increased the rate of proteolysis in the cheeses, and cheeses developed surface crystals and off-flavor at 12 wk of age. Addition of lyophilized protease and lipase enzymes derived from Aspergillus oryzae increased rates of proteolysis and lipolysis, but rancidity developed between 8 and 12 wk of age. The protease increased the rate of /3-casein hydrolysis. Condensed milk cheeses had more Cheddar flavor than the control cheese. (Key words: reduced fat, Cheddar cheese, proteolysis, accelerated ripening)
TS
= total
INTRODUCTION
Abbreviation key: ENZ = enzyme treatment, ET = elevated temperature treatment, MW = molecular weight, RFCC = reduced fat Ched-
Received April 19, 1993. Accepted December 14, 1993. 1Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 2729 of the Journal Series. 2Present address: Davisco International Inc., Lake Norden, SD 57248. 3Reprint requests (605/688-5731; FAX: 605/688-6065). 1994 J Dairy Sci 77:897-906
= treatment,
Problems related to body, texture, and flavor are common in reduced fat Cheddar cheese (RFCC) (12). The increased moisture content of RFCC can increase microbial populations and activity, which may produce excessive amounts of proteolytic enzymes that lead to off-flavors such as unclean and bitter (17). Because of low milk fat content in RFCC, relatively little Cheddar flavor from mixtures of alkanoic acids with carbon chains C2 to C8 or CIO is present in RFCC. However, the contribution of alkanoic acids to the aroma and special character of Cheddar cheese has not been proven (17). Cheese making requires high capital costs for plant and equipment and high fixed costs, such as insurance and taxes. Additionally, variable costs for utilities, labor, cheese storage, and initial raw materials are also incurred (22). The costs of manufacturing and aging cheese have therefore become more important. Accelerated ripening can help in quickly recovering the manufacturing cost of cheese. Cheese ripening can be accelerated in several ways, including elevated temperature (18), addition of enzymes (3, 10, 15, 16, 24), use of genetically modified starter bacteria (18), or use of adjunct cultures such as Micrococcus and Pediococcus spp. (7). In a previous publication (2), we reported on the potential use of condensed milk for making RFCC. Condensed milk at 1.8- to 2.0-fold concentration had the distinct advantage of producing good flavor with little bitterness and provided extra cheese yield. The objective of this study was to evaluate the use of flavor enzymes and a higher ripening temperature to accelerate flavor development in RFCC made from condensed milk.
897
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BRANDSMA ET AL.
MATERIALS AND METHODS Cheese Milk and Cheese Manufacture
Preparation of cheese milk and cheese manufacturing procedures were described in detail earlier (2): The four treatments (l'RT) were A, control, uncondensed; B, C, and D, condensed to approximately 1.5, 1.8, and 2.0 times the original total solids (1'8), respectively. The RFCC manufacturing characteristics, composition, yield, ripening, sensory evaluation, and microstructure were described in detail (1, 2). In the present study, the accelerated ripening of these cheeses was evaluated as follows: at milling, the curds from each of TRT A, B, C, and D were split into three ripening TRT: 1) control, ripened at 6 to 7·C, 2) elevated temperature (ET) TRT, ripened at II·C, and 3) enzyme (ENZ) TRT, ripened at 6 to TC, for a total of 12 TRT. Curd was salted at the rate of 2.5% by weight of the curd. For ENZ, a lyophilized commercial enzyme preparation (FlavorageFR®; Chr. Hansen's Lab., Inc., Milwaukee, WI) was mixed with dry salt and thoroughly mixed with the curd at the rate of .44 g of enzyme/IOO kg of wet curd. The enzyme preparation consisted of fungal lipase and protease and was derived from two strains of Aspergillus oryzae. Cheese curds (approximately 3.5 kg) were placed in forms and pressed (Kusel Equipment Co., Watertwon, WI) overnight (2.46 kg/cm2 ). Cheeses were sampled for composition within I wk after manufacture and evaluated for sensory characteristics and soluble nitrogen content at 0, 4, 8, and 12 wk of age. Proteolysis was also monitored by SDS-PAGE (6). Five replicates were conducted.
Sensory Evaluation
Randomly coded cheese samples were evaluated by a panel of five experienced judges at 0, 4, 8, and 12 wk of age. Samples were scored for flavor, texture, and appearance on a nine-point hedonic scale (1 = poor to 9 = excellent) and for Cheddar flavor intensity on a nine-point scale (1 = least intensity to 9 = highest intensity). Proteolysis
Acid-soluble nitrogen in the cheese was determined according to the method of Vakaleris and Price (26) and was calculated by using a standard curve derived by correlation with the Kjeldahl method for measurement of soluble protein in cheese (15). Proteolysis was also monitored with SDS-PAGE (6, 21) using a Bio-Rad Protean II Slab Cel1® (Bio-Rad, Richmond, CA). A 10 to 20% acrylamide gradient gel was employed. Gels were stained with .1 % Coomassie blue R250, 40% methanol, and 10% glacial acetic acid solution; destained in a 40% methanol and 10% glacial acetic acid solution; and dried in a Bio-Rad model 583 gel dryer. Gels were then scanned with a Bio-Rad model 620 video densitometer and analyzed using I-D Analyst software (Bio-Rad). Protein peaks were identified, and casein groups and breakdown products were calculated as percentages of total stained fractions (6). Three protein fractions (1, 2, and 3) were quantified. Fraction 1 consisted of a s l-, a s2-, and l3-caseins; fraction 2 was the intermediate proteolytic breakdown products between 13casein and para-K-casein; and fraction 3 contained proteolytic breakdown products with lower molecular weight (MW) than para-Kcasein (6). Statistical Analysis
Compositional Analysis
The macro-Kjeldahl method was used to determine total protein content of the cheese milk and cheese (4). Percentage of fat was determined by the Mojonnier method (4, 5), which was also used to determine TS in the cheese milk (4). Moisture content of cheese was determined using an Ohaus MB200 moisture balance (Ohaus Corp., Florham Park, NJ) (8), and salt in cheese was determined using a selective ion electrode (14). Journal of Dairy Science Vol. 77, No.4, 1994
Five replicates were conducted. Data were analyzed using the general linear models procedure from SAS (23); milk was the main plot, ripening conditions were the subplots, and ripening time was the sub-subplot (25). RESULTS Composition of Cheese
Milk and whey composition and cheese yield data were presented previously (1).
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REDUCED FAT CHEDDAR CHEESE
Cheese composltlon is presented in Table I. Moisture in RFCC from condensed milk decreased as TS concentration increased; the moisture content was different (P < .05) in all cheeses except those from TRT C and D. Cheese from TRT D had the highest (P < .05) total protein content. The major difference among the cheeses was the fat on a dry weight basis, which was highest (P < .05) in cheese from TRT A and lowest (P < .05) in cheese from TRT D (Table I). Sensory Evaluation
Flavor. At wk 0, flavor scores of cheeses made with TRT A and B were similar (P > .05) for all ripening TRT, as were the cheeses made with TRT C and D (Table 2). As the cheeses aged, the flavor score of the TRT A control cheese reached a peak at wk 8, but that of control cheeses made with TRT B, C, and D remained unchanged. By wk 8, flavor scores for the ET cheeses were lower than for the control cheeses for all concentrations, possibly because of different temperature optima for peptidases, which could lead to development of off-flavor (18). All ENZ cheeses developed rancidity; the TRT A ENZ cheese developed rancidity between 4 and 8 wk, and the TRT D ENZ cheese developed rancidity between 8 and 12 wk. Texture. Texture scores are presented in Table 3. The three ripening TRT had no significant effect on texture; hence, means across
all ripening TRT for the four milk concentrations are presented. Texture improved with age most markedly in the TRT A cheeses over 12 wk, but no significant improvement was observed in the cheeses from TRT C and D. At all stages of ripening, texture scores decreased with increasing milk concentration. Appearance. Effect of milk concentration on appearance was not significant (P > .05). The control cheeses had the highest (P < .05) appearance scores at 4, 8, and 12 wk, and ET cheeses had the lowest scores except at wk 4, when ET and ENZ cheeses were similar (Table 4).
With ET, calcium lactate crystallization became more pronounced than with other ripening TRT. Dybing et a1. (9) found little calcium lactate crystallization on Cheddar cheese stored at 12°C, but the most crystallization appeared on cheese stored at 6°C. Crystallization on the cheese surface was increased by the presence of gas-producing lactobacilli, which loosened the wrapping bag and promoted crystal formation (9, 13). Flavor Intensity. As expected, Cheddar flavor intensity scores increased over time across all milk concentrations for all ripening TRT (Table 5). Flavor intensity reached its peak at wk 8 for the cheeses from control and ET but continued to increase for 12 wk in ENZ cheeses. Flavor intensities for cheeses from ET and ENZ were similar until wk 4 (P > .05), but, at 8 and 12 wk, cheeses from ENZ had the highest score (P < .05).
TABLE 1. Composition! of reduced fat Cheddar cheese from condensed milk. Treatment 2 Component3
A
B
C
D
SE
17.6b 43.0" 32.5c 1.6b 30.9< 3.7" 52.2 c 5.lb
.19 .35 .21 .04 .36 .11 .38 .05
(%) Fat Moisture Total protein NaCI FDB S:M MFFC pH
IS.5·
46.oa 30.61.3· 34.2" 2.9< 56.4" 5.2"
IS.2·b 45.lb 31.2b 1.3" 33.l b 3.lbc 55.1" 5.Qb
IS.S" 43.3 c 31.3 b 1.5 b 33.l b 3.5 1b 53.3 b 5.Qb
l,b,cMeans in rows with like superscripts do not differ (p > .05). !Mean of five replicates.
=
2Treatments: A uncondensed milk (10.27% total solids (TS)]. B (18.33% TS), and D = condensed milk (22.23% TS).
=condensed milk (15.40% TS), C =condensed milk
3FDB = Fat on a dry weight basis, S:M = salt to moisture ratio. and MFFC
=moisture in fat-free cheese, pH at d
1.
Journal of Dairy Science Vol. 77, No.4. 1994
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BRANDSMA ET AL.
TABLE 2. Ravor scores l of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment2
o wk
4 wk
8 wk
12 wk
A Control ET ENZ
7.1 b,A 7.1 a.A 7.1a.A
7.2b.AB 7.4a.A 7.1 a,AB
7.S a.A 7.3 a.B 6.3 b.OEF
7.4ab .A 7.I I .AB 4.7 c•H
B Control ET ENZ
6.Sa.ABC 6.9"·AB 6.7a.ABC
6.S"·BC 6.3 b.OE 6.4"b.COE
6.S"·c 6.3 b.OEF 6.1 b.EFG
6.S"· BC 6.6 Ib .CO 5.3 c.G
C Control ET ENZ
6.6"· BC 6.4"·c 6.4"·c
6.S"·BC 6.5"·COE 6.5"·COE
6,6"· CO 6.5"· COE 5.8 b.G
6.8",BC 6.3"· OE 5.8 b.F
D Control ET ENZ
6.6"· BC 6.6a.DC 6.5·· DC
6.6"·CO 6.3· b.DE 6.1"b.E
6.3"· OEF 6.()b.FG 6.1"b.EFG
6.7"· BCO 5.9 b.EF 5.7 b.FG
a,b.cMeans in rows with like superscripts do not differ (P > .05). A.B,C.D.E.F.GMeans in columns within same milk concentration and age and with like superscripts do not differ (P > .05) 'Mean of five replicates and five judges; I
= poor
to 9
= excellent;
standard error of the mean
= .14.
2Treatments: A =uncondensed milk [10.27% total solids (IS)], B =condensed milk (15.40% TS), C =condensed milk (18.33% TS), and D = condensed milk (22.23% TS). Control ripening at 6 to TC, ET = elevated temperature ripening (ll'C), and ENZ = enzyme ripening (6 to 7'C).
Condensed milk cheeses from control and ET had higher flavor intensity scores than the cheeses from TRT A (P < .05) (fable 6). Cheeses from ENZ produced the highest flavor intensity in all cheeses (A, B, C, D), but, within the condensed milk cheeses, cheeses from TRT D for ENZ had the lowest flavor intensity scores.
Proteolysis
Acid-Soluble Nitrogen. The rate of proteolysis, as indicated by acid-soluble nitrogen, was lower in condensed milk cheeses than in the controls at 8 and 12 wk (P < .05) (Table 7). For all cheese milk concentrations, acid-soluble nitrogen increased during 12 wk of ripening (P
TABLE 3. Texture scores I of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment
o wk
4 wk
8 wk
12 wk
A B C D
5.2"·A 4.6·· B 4.oa· c 3.5". 0
6.Qb·A 4.9'b.B 4.5··B 3.7"·c
6.4 bc .A 5.lb.B 4.5"· c 3.9".0
6.8c.A 5.oab.B 4.3', c 3.7•. 0
a.b.CMeans in rows with like superscripts do not differ (P > .05). A,B.C.DMeans in columns with like superscripts do not differ (P > .05). IMeans across ripening treatments of five replicates and five judges; 1 mean = .17. 2Treatments: A =uncondensed milk (10,27% total solids (IS)], B (\8.33% TS), and D = condensed milk (22.23% TS). Journal of Dairy Science Vol. 77, No.4, 1994
=poor to 9 =excellent; standard error of the
=condensed milk (15.40% TS), C =condensed milk
901
REDUCED FAT CHEDDAR CHEESE TABLE 4. Appearance scores l for effect of ripening of reduced fat Cheddar cheese from condensed milk. Age of cheese
Ripening treatment 2
o wk
4 wk
8 wk
12 wk
Control ET ENZ
7.3 a.A 7.2 a,A 7.0a.A
7.5 a.A 6.7 b,B 7.1",B
7.4 a,A 6.3 c.B 6.8 a,c
6.8 b.A 5.6 d.B 6.Ib,C
a.b,cMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .05). lMeans across milk concentrations of five replicates and five judges; I mean = .14. 2Ripening treatments: control ripening at 6 to TC, ET ripening (6 to 7'C).
=poor to 9 = excellent; standard error of the
= elevated temperature
< .05); TRT 0 cheese exhibited the slowest rate of increase (P < .05). . Averaged across all ripening times, approximately 26% more soluble nitrogen was present in cheese from TRT A ENZ than in cheese from TRT A control, but only 14% more nitrogen was present with the TRT 0 ENZ cheese than in the TRT 0 control cheese (Table 8). An acceleration effect of 17% was caused by ET for the TRT A cheese compared with that of TRT A control cheese. This effect was lower (13%) for the TRT D ET cheese than for the TRT 0 control cheese. SDS-PAGE. A typical SDS-PAGE pattern for TRT A cheese at wk 12 (Figure 1) illustrates the differences between the three ripening TRT. The SDS-PAGE patterns for cheeses made with TRT B, C, and D were similar, except that breakdown products were fewer as concentration of cheese milk increased from TRT B to C to D. As a cheese ages, the concentrations of the caseins decrease, and concentrations of lower
ripening (lI'C). and ENZ
= enzyme
MW breakdown products of the caseins increase. These products appear in SDS-PAGE in the area between {1-casein and {1lactoglobulin (Figure 1) (6). Additionally, lower MW «14,000) products of proteolysis were also observed. All products, i.e., caseins and breakdown products, may be quantified and used to measure proteolysis of aging cheese (6).
Protein fractions identified through densitometry of the SDS-PAGE gels were classified into three groups (6) (Tables 9, 10, and II). Caseolysis was greatest in the TRT A ENZ cheese and least in the TRT D control cheese. Casein in TRT A ENZ decreased from 47.1 % of the total protein at the beginning of ripening to 18.7% after 12 wk; however, that in TRT A control decreased from 47.1 to 27.6%, which shows a 19% increase in caseolysis by ENZ (Table 9). Proteolysis of the casein fraction in TRT A ET cheeses was 13% higher than that of TRT A control. Among TRT B cheeses, ENZ increased casein proteol-
TABLE 5. Effect of ripening treatment on flavor intensity! scores of reduced fat Cheddar cheese from condensed milk. Age of cheese
Ripening treatment 2
o wk
4 wk
8 wk
12 wk
Control ET ENZ
4.2 a.A 4.2a.A 4.3 a.A
4.9b.A 5.1 b.B 5.2 b,B
5.7 c.A 6.0<·B 6.4c.c
5.6c.A 6.Ic.B 6.8d .C
a.b.c.dMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .OS). !Means across all milk concentrations of five replicates and five judges; I intensity; standard error of the mean = .1. 2Control ripening at 6 to TC, ET
= elevated temperature
=least flavor intensity to 9 = highest flavor
ripening (lI'C), and ENZ
= enzyme
ripening (6 to TC).
Journal of Dairy Science Vol. 77, No.4. 1994
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BRANDSMA ET AL
TABLE 6. Flavor intensity' scores of reduced fat Cheddar cheese from condensed milk, Treatment 2
Ripening treatment 3
A
B
C
D
Control ET ENZ
4.S a.A 5.0a.A 5.7 ab ,B
5.oac,A 5.6 b.B 5,sa.C
5,4 b.A 5,6 b.AB 5,S··B
5.lc.A 5.3 c.AIl 5.5 b.B
•.b,cMeans in rows with like superscripts do not differ (P > .05). A,B.cMeans in columns with like superscripts do not differ (P > .05), lMeans across all ages of five replicates and five judges; 1 = least flavor intensity to 9 = highest flavor intensity; standard error of the mean = .08. 2Treatments: A = uncondensed milk [10.27% total solids (TS)], B (lS.33% IS), and D = condensed milk (22.23% IS).
=condensed milk (15.40% IS), C = condensed milk
3Control ripening at 6 to TC, EI = elevated temperature ripening (11 "C), and ENZ = enzyme ripening (6 to TC).
ysis 11 % over the control ripening TRT, and ET increased proteolysis by 5.4%. For TRT 0 ENZ, proteolysis increased 15.7% over TRT 0 control, but ET increased proteolysis by only 2%.
The ENZ exhibited preference in the degradation of tl-casein, which has been implicated in producing bitterness (16), rather than the a scaseins. The cheeses from ENZ and control were similar in their degradation of the as· casein fractions, breaking down the as I-casein more quickly than the as2-casein, The data in Table 9 also show that most of the casein hydrolysis occurred between 0 and 8 wk of age; the rate of casein hydrolysis slowed sub· stantially (P < .05) between 8 and 12 wk. The second proteolysis group quantified was the group of protein peaks from tl-casein up to, but not including, para-K-casein (Table 10). This group consists of casein breakdown
products, which normally increase with age, but the concentration of the previous group decreases with age, For this group, milk concentration had no effect. The ENZ increased (P < .05) the percentage of these breakdown products because of the enzyme preference for tl-casein. After 12 wk of ripening, ENZ increased proteolysis by 61 %, but ET increased proteolysis 18% over that of the control. This difference suggests that the mode of protein hydrolysis with ENZ was different from that of the control and ET. The third protein group quantified was that of proteins with lower MW than para-K-casein (Table 11), which also increased as cheeses aged, After 12 wk of ripening, the acceleration effect of ENZ over control was 12%, but the increase with ET was 25%, averaged over all cheese milk concentrations. Cheeses of TRT 0 exhibited smaller proportions of these break-
TABLE 7. Acid-soluble nitrogen! in reduced fat Cheddar cheese from condensed milk across all ripening treatments. Age of cheese Treatment 2
o wk
4 wk
8 wk
12 wk
A B C D
.53 a,A .52a,A .54"A .53 a.A
.75 b,A .74 b,AIl .73 b,AB .69 b,B
1.03c,A .91 c,B ,S9c,B ,83 c,c
l.17 d,A 1,02d,1l I.ODd,B .97 d,B
.,b.c,dMeans in rows with like superscripts do not differ (P > .05). A.B,cMeans in columns with like superscripts do not differ (P > .05). 1Means
across ripening treatments of five replicates; standard error of the mean = .02.
2Ireatments: A = uncondensed milk [10.27% total solids (TS)], B (1833% IS), and D = condensed milk (22.23% IS). Journal of Dairy Science Vol. 77, No.4, 1994
=condensed milk (15.40% IS), C =condensed milk
903
REDUCED FAT CHEDDAR CHEESE
down products at 4, 8, and 12 wk than did TRT A cheeses.
lipolysis. An increase in proteinase addition may promote a faster rate of desired texture development, especially with slower ripening cheeses. Analysis of cheese samples by SDS-PAGE showed definite proteolytic effects of concentration of cheese milk and ripening TRT. Protein groups quantified through the use of densitometry showed differences in proteolytic activity, each greater than the next in the order ENZ, ET, control, and TRT A, B, C, and D (P < .05) (Tables 9, 10, and II). Several important variables influence cheese ripening, including the salt-to-moisture ratio, milk quality, ripening temperature, bacterial count and species involved, residual coagulant in the cheese curd, and the effects of different manufacturing procedures (15, 19). Bacterial growth factors include the salt and moisture content, pH at milling, cooking temperature, and populations of nonstarter lactic acid bacteria, which may be added to increase the ripening rate. Increased proteolytic activity in ENZ and ET cheeses, indicated by increased amounts of
DISCUSSION
All cheeses produced had at least a onethird reduction in fat content (dry weight basis) and :S;125% of the maximum allowable moisture content of the standard cheese variety. In a preliminary experiment, equal amounts of the commercial enzyme preparation were used for preparation of full fat and RFCC and ripened at 6 to 7°C. The full fat Cheddar cheese had excellent flavor at 9 wk, but the RFCC had developed rancidity. At 16 wk, the RFCC was rancid, but the full fat cheese maintained good flavor and texture development. Hargrove et al. (11) suggested that low fat cheeses with added enz.yme produce extensive rancidity. Milk fat may also play the role of masking off-flavor in cheese (22). This role of milk fat may not be fully available in RFCC. Thus, with a reduction in fat content in the cheese milk, the rate of lipase addition would also have to be reduced to prevent excessive
MW 97,400 66,200 45,000 31,000 21,500 14,400
u s2 -CN -----us,-CN ----.:. B-CN--
B-LG _ _ para-K-CN___
u-LA _ _
Control
ET
ENZ
Figure 1. The SDS-PAGE lanes of reduced fat Cheddar cheese from treatment A control uncondensed milk, 12 wk of age. Lane I, low molecular weight (MW) standard containing phosphorylase b (MW 97,400), BSA (MW 66,200), ovalbumin (MW 45,000), bovine carbonic anhydrase (MW 31,(00), soybean trypsin inhibitor (MW 21,500), and egg white lysozyme (MW 14,400). Treatments: control ripening (6 to 7"C), ET = elevated temperature ripening (II "C), ENZ = enzyme ripening (6 to 7'C). Journal of Dairy Science Vol. 77, No.4, 1994
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BRANDSMA ET AL.
TABLE 8. Acid-soluble nitrogen 1 in reduced fat Cheddar cheese from condensed milk across all ages. Treatment 2
Ripening treatment 3
A
B
C
D
Conlrol ET ENZ
.76a.A .89 a.B .96a.C
.74 ab .A .82 b.B .84b.B
.72 bc •A .82 b.B .84b.B
.69 c.A .78 c.B .79 d.B
a.b.CMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .05). !Means across ripening treatments of five replicates; standard error of the mean
= .01.
2Treatments: A = uncondensed milk [10.27% total solids (TS)], B = condensed milk (15.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% TS). 3Control ripening at 6 to 7'C, ET
= elevated temperature
soluble nitrogen, did not improve cheese texture, perhaps because of insufficient proteolysis or because of the nature of proteolytic breakdown products. The retardation of ripening was likely due to compositional imbalances in the condensed milk cheeses, such as higher mineral contents, which decreased bacterial activity (2) as the concentration of cheese milks increased.
ripening (I I 'C), and ENZ
= enzyme
ripening (6 to TC).
Temperature also determines the rate of ripening; the rate of flavor and texture development is influenced by proteolytic and lipolytic activities of bacterial enzymes. The optimal temperature may be higher to allow for maximal proteolytic effect of the enzyme because this effect also occurred in RFCC from UF milk concentrated five times (20). Elevated temperatures to accelerate the rate of
TABLE 9. Specific casein fraction percentages! (0<51-. 0<52-' and i3-caseins) of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment2
o wk
4 wk
8 wk
12 wk
(%) A Control ET ENZ
47.J&·A 47.J&·A 47. J&.A
36.7 b.BC 35.3 b.C 30.7 b.E
30.3c.CD 25.7 c.G 22.OC· H
27.6 d.CD 24.9c.FG 18.7d.H
B Control ET ENZ
43.9 a.B 43.9 a.B 43.9 a.B
36.1 b.BC 34.9 b.C 32.6 b.D
30.OC· CD 28.6 c.DE 27.9c.EF
28.4c.c 26.Qd·DEF 23.5 d .G
C Control ET ENZ
45.4a.AB 45.4·· AB 45.4··AB
36.2 b.BC 35.0 b.C 32.7 b.D
33.4c.B 29.5 c.CDE 26.2c.FG
32.lc.B 28.OC·c 25.4c.EF
D Control ET ENZ
45.2·· B 45.2··B 45.2·· B
38.5 b.A 37.1 MB 36.4b.BC
35.2c.A 34.1 c.AB 30.6c.C
33.9c.A 32.9c.A 26.8 d.CDE
a.b.c.dMeans in rows with like superscripts do not differ (P > .05). A.B.C.D.E.FMeans in columns with like superscripts within same milk concentration and age do not differ (P > .05). I Mean
of five replicates; standard error of the mean
= .62.
2Treatments: A = uncondensed milk [10.27% total solids (TS)]. B = condensed milk (15.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% TS). Control ripening at 6 to 7'C. ET = elevated temperature ripening (11 T). and ENZ = enzyme ripening (6 to TC). Journal of Dairy Science Vol. 77. No.4. 1994
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REDUCED FAT CHEDDAR CHEESE
TABLE 10. Specific protein breakdown percentages' (components between l3-casein and para-K-casein in SDS-PAGE) of reduced fat Cheddar cheese from condensed milk. Ripening treatment 2
Age of cheese
o wk
4 wk
8 wk
12 wk
2S.2c.A 27.7 c,B 31.2c,c
28.9C·A 30.8d,B 3S.3d,c
(%) Control ET ENZ
18.4··A 18.4··B 18.4',c
22.2 b,A 23.7 b.B 26.lb.C
•.b.c.dMeans in rows with like superscripts do not differ (P > .OS). A,B.cMeans in columns with like superscripts do not differ (P > .OS). IMeans across all milk concentrations of five replicates; standard error of the mean
= .39.
2Control ripening at 6 to TC, ET = elevated temperature ripening (l1"C), and ENZ = enzyme ripening (6 to TC).
ripening must be used with caution; off-flavor development can occur from uncontrolled proteolysis, The condensed milk cheese generally had a good Cheddar flavor that was stronger than that of the uncondensed milk cheese, but overall flavor scores of the condensed milk control cheeses were lower than those of the uncondensed control. The increased yield and longer shelf-life than most commercial RFCC are advantages of condensed milk (2). Also, less lipolytic activity is needed in RFCC to achieve an acceleration effect of the aroma and taste. Further study of protease addition to RFCC is needed.
The high moisture content in cheese from A ET led to off-flavor development from uncontrolled proteolysis, With ET cheeses from condensed milk, the higher mineral content led to excessive moisture expulsion and a dry, short texture with some off-flavor development in addition to slower ripening. Basic compositional problems of the cheese milk and cheesemaking procedures need to be resolved before elevated temperatures are used to ripen for acceleration of the cheese maturation process, Treatment of RFCC with enzymes is a viable alternative to accelerate the ripening process, but more research is needed to determine
TABLE II. Specific protein breakdown percentages (lower molecular weight than para-K-casein) of reduced fat Cheddar cheese from condensed milk. Age
o wk
4 wk
8 wk
12 wk
(%) Treatments I A B C D Ripening treatment 2 Control ET ENZ
8.6"A 1O.lc.8 IO.S·,8 9.4·· AB
14.4b.A 14.8b,A IS.Ob,A 12.S b,B
17.sc,A 16.7c,A 16.2b.A B.Sb,8
20.2d,A 17.8c.8 18.6c.8 16.OC·c
9.6·,A 9.6"A 9.6·,A
14.0b,A 14.Sb,A 14.0b,A
IS.3 c,A 16.6c,B lS.9c,AB
17.2d,A 19.1d,B 18.1 d,C
•.b.c.dMeans in rows with like superscripts do not differ (P > .OS). A.B,cMeans in columns with like superscripts within concentration or ripening treatment do not differ (P > .OS). IMeans across all ripening treatments of five replicates. Treatments: A = uncondensed milk [10.27% total solids (TS)], B = condensed milk (1S.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% IS); standard error of the mean = .47. 2Means across all milk concentrations of five replicates. Control ripening at 6 to 7"C, ET = elevated temperature ripening (I I"C), and ENZ = enzyme ripening (6 to TC); standard error = .23. Journal of Dairy Science Vol. 77, No.4, 1994
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BRANDSMA ET AL
the best sources of enzymes and the correct blend of proteases for texture development. CONCLUSIONS
Accelerated ripening is an alternative method for flavor development in RFCC from condensed milk. The RFCC from condensed milk had firm, crumbly body but had a higher Cheddar flavor score than control cheeses. The rate of ripening, as indicated by soluble nitrogen and electrophoresis, was slower in the condensed milk cheeses than in the control cheeses. Rancid flavor developed rapidly in all cheeses made with added enzyme; hence, enzyme preparations with balanced proteases and lipases should be selected. ACKNOWLEDGMENTS
The authors thank the Minnesota-South Dakota Dairy Foods Research Center for funding this project and W. Lee Tucker, South Dakota State University, for providing valuable help with statistical analysis. REFERENCES
1 Anderson. D. L. and V. V. Mistry. 1994. Reduced fat Cheddar cheese from condensed milk. 2. Microstructure. 1. Dairy Sci. 77:7. 2 Anderson. D. L. V. V. Mistry. R. L Brandsma, and K. A. Baldwin. 1993. Reduced fat Cheddar cheese from condensed milk. 1. Manufacture, composition, and yield. 1. Dairy Sci. 76:2832. 3 Arbige, M. V., P. R Freund, S. C. Silver, and J. T. Zelko. 1986. Novel lipase for Cheddar cheese flavor development. Food Techno\. 40:91. 4 Association of Official Analytical Chemists. 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. 5 Atherton, H. V., and J. A. Newlander. 1977. Chemistry and Testing of Dairy Products. 4th ed. AVI Pub\. Co. Inc., Westport, CT. 6 Basch. J. 1., H. M. Farrell, Jr., R. A. Walsh, R. P, Konstance, and T. F. Kumosinski. 1989. Development of a quantitative model for enzyme-catalyzed. time dependent changes in protein composition of Cheddar cheese during storage. J. Dairy Sci. 72:591. 7 Bhowmik, T., and E. H. Marth, 1990. Role of Micrococcus and Pediococcus species in cheese ripening. 1. Dairy Sci. 73:859. 8 Crosser, A. E., and V. V. Mistry. 1991. Use of a moisture balance to determine moisture in cheese. 1. Dairy Sci. 74(Suppl I): 126.(Abstr.)
10urnal of Dairy Science Vo\. 77, No.4, 1994
9 Dybing, S. T., 1. A. Wiegand, S. A. Brudvig, E. A. Huang, and R. C. Chandan. 1988, Effect of processing variables on the formation of calcium lactate crystals on Cheddar cheese, J. Dairy Sci. 71:1701. 10 EI Soda, M. E.. and S. Pandian. 1991. Recent developments in accelerated cheese ripening. J. Dairy Sci. 74: 2317. 11 Hargrove, R. E., F. E. McDonough, and R. P. Tittsler. 1967. Factors affecting characteristics, composition and quality of skim milk cheese. J. Dairy Sci, 50:160. 12 Jameson. G. W. 1990. Cheese with less fat. Aust. J. Dairy Techno\. 45:93. 13 Johnson, M. E., B. A. Riesterer, and N. F. Olson, 1990. Influence of nonstarter bacteria on calcium lactate crystallization on the surface of Cheddar cheese. J. Dairy Sci. 73:1145. 14 Kindstedt, P. A., and F. V. Kosikowski. 1984. Measurement of sodium chloride in cheese by a simple sodium ion electrode method. 1. Dairy Sci. 67:879. 15 Kosikowski, F. V. 1982. Cheese and Fermented Milk Foods. 2nd ed, F. V. Kosikowski and Assoc., Brooktondale, NY. 16 Law. B. A. 1981. Accelerated ripening of Cheddar cheese with microbial proteinases. Neth. Milk Dairy J. 35:313. 17 Law. B, A. 1984. Flavor development in cheese. Page 187 in Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. F. L Davies and B. A. Law, ed. Elsevier App\. Sci. Pub\., London, Engl. 18 Law, B. A. 1984. The accelerated ripening of cheese, Page 209 in Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk. F. L Davies and B, A. Law, ed. Elsevier App\. Sci. Publ., London, Eng\. 19 Lawrence, R. C., L K. Creamer, and 1. Gilles. 1987. Texture development during cheese ripening, J. Dairy Sci. 70:1748. 20 McGregor, J. U., and C. H. White. 1990. Effect of enzyme treatment and ultrafiltration on the quality of low fat Cheddar cheese. J. Dairy Sci. 73:571. 21 Mistry, V. V., and N, H. Hassan, 1991. Delactosed, high milk protein powder. 1. Manufacture and composition. 1. Dairy Sci. 74:1163. 22 Olson, N. F., and M. E. Johnson. 1990. Light cheese products: characteristics and economics. Food Technol. 44(10):93. 23 SAS® User's Guide: Statistics, Version 6.0 Edition. 1990. SAS Inst., Inc., Cary, NC. 24 Sood, V. K., and F. V. Kosikowski. 1979. Accelerated Cheddar cheese ripening by added microbial enzymes. J. Dairy Sci. 62:1865. 25 Steele, RG.D., and 1. H. Torrie. 1980. Principles and Procedures of Statistics: A Biornetrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY. 26 Vakaleris, D. G" and W. V. Price. 1959. A rapid spectrophotometric method for measuring cheese ripening. 1. Dairy Sci. 42:264.
dar cheese, TRT solids.
Reduced fat Cheddar cheeses were manufactured from control uncondensed milk (10.27% total solids) and condensed milks containing 15.40, 18.33, and 22.23% total solids. Three ripening treatments were applied to the cheeses: control, 6 to 7°C; elevated temperature, 11·C; and enzyme added, 6 to 7·C. The cheeses were ripened for up to 12 wk. Soluble nitrogen production was slower in the condensed milk cheeses. Condensed milk cheeses were drier and had crumbly body with increasing milk concentration. Elevated ripening temperatures increased the rate of proteolysis in the cheeses, and cheeses developed surface crystals and off-flavor at 12 wk of age. Addition of lyophilized protease and lipase enzymes derived from Aspergillus oryzae increased rates of proteolysis and lipolysis, but rancidity developed between 8 and 12 wk of age. The protease increased the rate of /3-casein hydrolysis. Condensed milk cheeses had more Cheddar flavor than the control cheese. (Key words: reduced fat, Cheddar cheese, proteolysis, accelerated ripening)
TS
= total
INTRODUCTION
Abbreviation key: ENZ = enzyme treatment, ET = elevated temperature treatment, MW = molecular weight, RFCC = reduced fat Ched-
Received April 19, 1993. Accepted December 14, 1993. 1Published with the approval of the director of the South Dakota Agricultural Experiment Station as Publication Number 2729 of the Journal Series. 2Present address: Davisco International Inc., Lake Norden, SD 57248. 3Reprint requests (605/688-5731; FAX: 605/688-6065). 1994 J Dairy Sci 77:897-906
= treatment,
Problems related to body, texture, and flavor are common in reduced fat Cheddar cheese (RFCC) (12). The increased moisture content of RFCC can increase microbial populations and activity, which may produce excessive amounts of proteolytic enzymes that lead to off-flavors such as unclean and bitter (17). Because of low milk fat content in RFCC, relatively little Cheddar flavor from mixtures of alkanoic acids with carbon chains C2 to C8 or CIO is present in RFCC. However, the contribution of alkanoic acids to the aroma and special character of Cheddar cheese has not been proven (17). Cheese making requires high capital costs for plant and equipment and high fixed costs, such as insurance and taxes. Additionally, variable costs for utilities, labor, cheese storage, and initial raw materials are also incurred (22). The costs of manufacturing and aging cheese have therefore become more important. Accelerated ripening can help in quickly recovering the manufacturing cost of cheese. Cheese ripening can be accelerated in several ways, including elevated temperature (18), addition of enzymes (3, 10, 15, 16, 24), use of genetically modified starter bacteria (18), or use of adjunct cultures such as Micrococcus and Pediococcus spp. (7). In a previous publication (2), we reported on the potential use of condensed milk for making RFCC. Condensed milk at 1.8- to 2.0-fold concentration had the distinct advantage of producing good flavor with little bitterness and provided extra cheese yield. The objective of this study was to evaluate the use of flavor enzymes and a higher ripening temperature to accelerate flavor development in RFCC made from condensed milk.
897
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BRANDSMA ET AL.
MATERIALS AND METHODS Cheese Milk and Cheese Manufacture
Preparation of cheese milk and cheese manufacturing procedures were described in detail earlier (2): The four treatments (l'RT) were A, control, uncondensed; B, C, and D, condensed to approximately 1.5, 1.8, and 2.0 times the original total solids (1'8), respectively. The RFCC manufacturing characteristics, composition, yield, ripening, sensory evaluation, and microstructure were described in detail (1, 2). In the present study, the accelerated ripening of these cheeses was evaluated as follows: at milling, the curds from each of TRT A, B, C, and D were split into three ripening TRT: 1) control, ripened at 6 to 7·C, 2) elevated temperature (ET) TRT, ripened at II·C, and 3) enzyme (ENZ) TRT, ripened at 6 to TC, for a total of 12 TRT. Curd was salted at the rate of 2.5% by weight of the curd. For ENZ, a lyophilized commercial enzyme preparation (FlavorageFR®; Chr. Hansen's Lab., Inc., Milwaukee, WI) was mixed with dry salt and thoroughly mixed with the curd at the rate of .44 g of enzyme/IOO kg of wet curd. The enzyme preparation consisted of fungal lipase and protease and was derived from two strains of Aspergillus oryzae. Cheese curds (approximately 3.5 kg) were placed in forms and pressed (Kusel Equipment Co., Watertwon, WI) overnight (2.46 kg/cm2 ). Cheeses were sampled for composition within I wk after manufacture and evaluated for sensory characteristics and soluble nitrogen content at 0, 4, 8, and 12 wk of age. Proteolysis was also monitored by SDS-PAGE (6). Five replicates were conducted.
Sensory Evaluation
Randomly coded cheese samples were evaluated by a panel of five experienced judges at 0, 4, 8, and 12 wk of age. Samples were scored for flavor, texture, and appearance on a nine-point hedonic scale (1 = poor to 9 = excellent) and for Cheddar flavor intensity on a nine-point scale (1 = least intensity to 9 = highest intensity). Proteolysis
Acid-soluble nitrogen in the cheese was determined according to the method of Vakaleris and Price (26) and was calculated by using a standard curve derived by correlation with the Kjeldahl method for measurement of soluble protein in cheese (15). Proteolysis was also monitored with SDS-PAGE (6, 21) using a Bio-Rad Protean II Slab Cel1® (Bio-Rad, Richmond, CA). A 10 to 20% acrylamide gradient gel was employed. Gels were stained with .1 % Coomassie blue R250, 40% methanol, and 10% glacial acetic acid solution; destained in a 40% methanol and 10% glacial acetic acid solution; and dried in a Bio-Rad model 583 gel dryer. Gels were then scanned with a Bio-Rad model 620 video densitometer and analyzed using I-D Analyst software (Bio-Rad). Protein peaks were identified, and casein groups and breakdown products were calculated as percentages of total stained fractions (6). Three protein fractions (1, 2, and 3) were quantified. Fraction 1 consisted of a s l-, a s2-, and l3-caseins; fraction 2 was the intermediate proteolytic breakdown products between 13casein and para-K-casein; and fraction 3 contained proteolytic breakdown products with lower molecular weight (MW) than para-Kcasein (6). Statistical Analysis
Compositional Analysis
The macro-Kjeldahl method was used to determine total protein content of the cheese milk and cheese (4). Percentage of fat was determined by the Mojonnier method (4, 5), which was also used to determine TS in the cheese milk (4). Moisture content of cheese was determined using an Ohaus MB200 moisture balance (Ohaus Corp., Florham Park, NJ) (8), and salt in cheese was determined using a selective ion electrode (14). Journal of Dairy Science Vol. 77, No.4, 1994
Five replicates were conducted. Data were analyzed using the general linear models procedure from SAS (23); milk was the main plot, ripening conditions were the subplots, and ripening time was the sub-subplot (25). RESULTS Composition of Cheese
Milk and whey composition and cheese yield data were presented previously (1).
899
REDUCED FAT CHEDDAR CHEESE
Cheese composltlon is presented in Table I. Moisture in RFCC from condensed milk decreased as TS concentration increased; the moisture content was different (P < .05) in all cheeses except those from TRT C and D. Cheese from TRT D had the highest (P < .05) total protein content. The major difference among the cheeses was the fat on a dry weight basis, which was highest (P < .05) in cheese from TRT A and lowest (P < .05) in cheese from TRT D (Table I). Sensory Evaluation
Flavor. At wk 0, flavor scores of cheeses made with TRT A and B were similar (P > .05) for all ripening TRT, as were the cheeses made with TRT C and D (Table 2). As the cheeses aged, the flavor score of the TRT A control cheese reached a peak at wk 8, but that of control cheeses made with TRT B, C, and D remained unchanged. By wk 8, flavor scores for the ET cheeses were lower than for the control cheeses for all concentrations, possibly because of different temperature optima for peptidases, which could lead to development of off-flavor (18). All ENZ cheeses developed rancidity; the TRT A ENZ cheese developed rancidity between 4 and 8 wk, and the TRT D ENZ cheese developed rancidity between 8 and 12 wk. Texture. Texture scores are presented in Table 3. The three ripening TRT had no significant effect on texture; hence, means across
all ripening TRT for the four milk concentrations are presented. Texture improved with age most markedly in the TRT A cheeses over 12 wk, but no significant improvement was observed in the cheeses from TRT C and D. At all stages of ripening, texture scores decreased with increasing milk concentration. Appearance. Effect of milk concentration on appearance was not significant (P > .05). The control cheeses had the highest (P < .05) appearance scores at 4, 8, and 12 wk, and ET cheeses had the lowest scores except at wk 4, when ET and ENZ cheeses were similar (Table 4).
With ET, calcium lactate crystallization became more pronounced than with other ripening TRT. Dybing et a1. (9) found little calcium lactate crystallization on Cheddar cheese stored at 12°C, but the most crystallization appeared on cheese stored at 6°C. Crystallization on the cheese surface was increased by the presence of gas-producing lactobacilli, which loosened the wrapping bag and promoted crystal formation (9, 13). Flavor Intensity. As expected, Cheddar flavor intensity scores increased over time across all milk concentrations for all ripening TRT (Table 5). Flavor intensity reached its peak at wk 8 for the cheeses from control and ET but continued to increase for 12 wk in ENZ cheeses. Flavor intensities for cheeses from ET and ENZ were similar until wk 4 (P > .05), but, at 8 and 12 wk, cheeses from ENZ had the highest score (P < .05).
TABLE 1. Composition! of reduced fat Cheddar cheese from condensed milk. Treatment 2 Component3
A
B
C
D
SE
17.6b 43.0" 32.5c 1.6b 30.9< 3.7" 52.2 c 5.lb
.19 .35 .21 .04 .36 .11 .38 .05
(%) Fat Moisture Total protein NaCI FDB S:M MFFC pH
IS.5·
46.oa 30.61.3· 34.2" 2.9< 56.4" 5.2"
IS.2·b 45.lb 31.2b 1.3" 33.l b 3.lbc 55.1" 5.Qb
IS.S" 43.3 c 31.3 b 1.5 b 33.l b 3.5 1b 53.3 b 5.Qb
l,b,cMeans in rows with like superscripts do not differ (p > .05). !Mean of five replicates.
=
2Treatments: A uncondensed milk (10.27% total solids (TS)]. B (18.33% TS), and D = condensed milk (22.23% TS).
=condensed milk (15.40% TS), C =condensed milk
3FDB = Fat on a dry weight basis, S:M = salt to moisture ratio. and MFFC
=moisture in fat-free cheese, pH at d
1.
Journal of Dairy Science Vol. 77, No.4. 1994
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BRANDSMA ET AL.
TABLE 2. Ravor scores l of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment2
o wk
4 wk
8 wk
12 wk
A Control ET ENZ
7.1 b,A 7.1 a.A 7.1a.A
7.2b.AB 7.4a.A 7.1 a,AB
7.S a.A 7.3 a.B 6.3 b.OEF
7.4ab .A 7.I I .AB 4.7 c•H
B Control ET ENZ
6.Sa.ABC 6.9"·AB 6.7a.ABC
6.S"·BC 6.3 b.OE 6.4"b.COE
6.S"·c 6.3 b.OEF 6.1 b.EFG
6.S"· BC 6.6 Ib .CO 5.3 c.G
C Control ET ENZ
6.6"· BC 6.4"·c 6.4"·c
6.S"·BC 6.5"·COE 6.5"·COE
6,6"· CO 6.5"· COE 5.8 b.G
6.8",BC 6.3"· OE 5.8 b.F
D Control ET ENZ
6.6"· BC 6.6a.DC 6.5·· DC
6.6"·CO 6.3· b.DE 6.1"b.E
6.3"· OEF 6.()b.FG 6.1"b.EFG
6.7"· BCO 5.9 b.EF 5.7 b.FG
a,b.cMeans in rows with like superscripts do not differ (P > .05). A.B,C.D.E.F.GMeans in columns within same milk concentration and age and with like superscripts do not differ (P > .05) 'Mean of five replicates and five judges; I
= poor
to 9
= excellent;
standard error of the mean
= .14.
2Treatments: A =uncondensed milk [10.27% total solids (IS)], B =condensed milk (15.40% TS), C =condensed milk (18.33% TS), and D = condensed milk (22.23% TS). Control ripening at 6 to TC, ET = elevated temperature ripening (ll'C), and ENZ = enzyme ripening (6 to 7'C).
Condensed milk cheeses from control and ET had higher flavor intensity scores than the cheeses from TRT A (P < .05) (fable 6). Cheeses from ENZ produced the highest flavor intensity in all cheeses (A, B, C, D), but, within the condensed milk cheeses, cheeses from TRT D for ENZ had the lowest flavor intensity scores.
Proteolysis
Acid-Soluble Nitrogen. The rate of proteolysis, as indicated by acid-soluble nitrogen, was lower in condensed milk cheeses than in the controls at 8 and 12 wk (P < .05) (Table 7). For all cheese milk concentrations, acid-soluble nitrogen increased during 12 wk of ripening (P
TABLE 3. Texture scores I of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment
o wk
4 wk
8 wk
12 wk
A B C D
5.2"·A 4.6·· B 4.oa· c 3.5". 0
6.Qb·A 4.9'b.B 4.5··B 3.7"·c
6.4 bc .A 5.lb.B 4.5"· c 3.9".0
6.8c.A 5.oab.B 4.3', c 3.7•. 0
a.b.CMeans in rows with like superscripts do not differ (P > .05). A,B.C.DMeans in columns with like superscripts do not differ (P > .05). IMeans across ripening treatments of five replicates and five judges; 1 mean = .17. 2Treatments: A =uncondensed milk (10,27% total solids (IS)], B (\8.33% TS), and D = condensed milk (22.23% TS). Journal of Dairy Science Vol. 77, No.4, 1994
=poor to 9 =excellent; standard error of the
=condensed milk (15.40% TS), C =condensed milk
901
REDUCED FAT CHEDDAR CHEESE TABLE 4. Appearance scores l for effect of ripening of reduced fat Cheddar cheese from condensed milk. Age of cheese
Ripening treatment 2
o wk
4 wk
8 wk
12 wk
Control ET ENZ
7.3 a.A 7.2 a,A 7.0a.A
7.5 a.A 6.7 b,B 7.1",B
7.4 a,A 6.3 c.B 6.8 a,c
6.8 b.A 5.6 d.B 6.Ib,C
a.b,cMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .05). lMeans across milk concentrations of five replicates and five judges; I mean = .14. 2Ripening treatments: control ripening at 6 to TC, ET ripening (6 to 7'C).
=poor to 9 = excellent; standard error of the
= elevated temperature
< .05); TRT 0 cheese exhibited the slowest rate of increase (P < .05). . Averaged across all ripening times, approximately 26% more soluble nitrogen was present in cheese from TRT A ENZ than in cheese from TRT A control, but only 14% more nitrogen was present with the TRT 0 ENZ cheese than in the TRT 0 control cheese (Table 8). An acceleration effect of 17% was caused by ET for the TRT A cheese compared with that of TRT A control cheese. This effect was lower (13%) for the TRT D ET cheese than for the TRT 0 control cheese. SDS-PAGE. A typical SDS-PAGE pattern for TRT A cheese at wk 12 (Figure 1) illustrates the differences between the three ripening TRT. The SDS-PAGE patterns for cheeses made with TRT B, C, and D were similar, except that breakdown products were fewer as concentration of cheese milk increased from TRT B to C to D. As a cheese ages, the concentrations of the caseins decrease, and concentrations of lower
ripening (lI'C). and ENZ
= enzyme
MW breakdown products of the caseins increase. These products appear in SDS-PAGE in the area between {1-casein and {1lactoglobulin (Figure 1) (6). Additionally, lower MW «14,000) products of proteolysis were also observed. All products, i.e., caseins and breakdown products, may be quantified and used to measure proteolysis of aging cheese (6).
Protein fractions identified through densitometry of the SDS-PAGE gels were classified into three groups (6) (Tables 9, 10, and II). Caseolysis was greatest in the TRT A ENZ cheese and least in the TRT D control cheese. Casein in TRT A ENZ decreased from 47.1 % of the total protein at the beginning of ripening to 18.7% after 12 wk; however, that in TRT A control decreased from 47.1 to 27.6%, which shows a 19% increase in caseolysis by ENZ (Table 9). Proteolysis of the casein fraction in TRT A ET cheeses was 13% higher than that of TRT A control. Among TRT B cheeses, ENZ increased casein proteol-
TABLE 5. Effect of ripening treatment on flavor intensity! scores of reduced fat Cheddar cheese from condensed milk. Age of cheese
Ripening treatment 2
o wk
4 wk
8 wk
12 wk
Control ET ENZ
4.2 a.A 4.2a.A 4.3 a.A
4.9b.A 5.1 b.B 5.2 b,B
5.7 c.A 6.0<·B 6.4c.c
5.6c.A 6.Ic.B 6.8d .C
a.b.c.dMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .OS). !Means across all milk concentrations of five replicates and five judges; I intensity; standard error of the mean = .1. 2Control ripening at 6 to TC, ET
= elevated temperature
=least flavor intensity to 9 = highest flavor
ripening (lI'C), and ENZ
= enzyme
ripening (6 to TC).
Journal of Dairy Science Vol. 77, No.4. 1994
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BRANDSMA ET AL
TABLE 6. Flavor intensity' scores of reduced fat Cheddar cheese from condensed milk, Treatment 2
Ripening treatment 3
A
B
C
D
Control ET ENZ
4.S a.A 5.0a.A 5.7 ab ,B
5.oac,A 5.6 b.B 5,sa.C
5,4 b.A 5,6 b.AB 5,S··B
5.lc.A 5.3 c.AIl 5.5 b.B
•.b,cMeans in rows with like superscripts do not differ (P > .05). A,B.cMeans in columns with like superscripts do not differ (P > .05), lMeans across all ages of five replicates and five judges; 1 = least flavor intensity to 9 = highest flavor intensity; standard error of the mean = .08. 2Treatments: A = uncondensed milk [10.27% total solids (TS)], B (lS.33% IS), and D = condensed milk (22.23% IS).
=condensed milk (15.40% IS), C = condensed milk
3Control ripening at 6 to TC, EI = elevated temperature ripening (11 "C), and ENZ = enzyme ripening (6 to TC).
ysis 11 % over the control ripening TRT, and ET increased proteolysis by 5.4%. For TRT 0 ENZ, proteolysis increased 15.7% over TRT 0 control, but ET increased proteolysis by only 2%.
The ENZ exhibited preference in the degradation of tl-casein, which has been implicated in producing bitterness (16), rather than the a scaseins. The cheeses from ENZ and control were similar in their degradation of the as· casein fractions, breaking down the as I-casein more quickly than the as2-casein, The data in Table 9 also show that most of the casein hydrolysis occurred between 0 and 8 wk of age; the rate of casein hydrolysis slowed sub· stantially (P < .05) between 8 and 12 wk. The second proteolysis group quantified was the group of protein peaks from tl-casein up to, but not including, para-K-casein (Table 10). This group consists of casein breakdown
products, which normally increase with age, but the concentration of the previous group decreases with age, For this group, milk concentration had no effect. The ENZ increased (P < .05) the percentage of these breakdown products because of the enzyme preference for tl-casein. After 12 wk of ripening, ENZ increased proteolysis by 61 %, but ET increased proteolysis 18% over that of the control. This difference suggests that the mode of protein hydrolysis with ENZ was different from that of the control and ET. The third protein group quantified was that of proteins with lower MW than para-K-casein (Table 11), which also increased as cheeses aged, After 12 wk of ripening, the acceleration effect of ENZ over control was 12%, but the increase with ET was 25%, averaged over all cheese milk concentrations. Cheeses of TRT 0 exhibited smaller proportions of these break-
TABLE 7. Acid-soluble nitrogen! in reduced fat Cheddar cheese from condensed milk across all ripening treatments. Age of cheese Treatment 2
o wk
4 wk
8 wk
12 wk
A B C D
.53 a,A .52a,A .54"A .53 a.A
.75 b,A .74 b,AIl .73 b,AB .69 b,B
1.03c,A .91 c,B ,S9c,B ,83 c,c
l.17 d,A 1,02d,1l I.ODd,B .97 d,B
.,b.c,dMeans in rows with like superscripts do not differ (P > .05). A.B,cMeans in columns with like superscripts do not differ (P > .05). 1Means
across ripening treatments of five replicates; standard error of the mean = .02.
2Ireatments: A = uncondensed milk [10.27% total solids (TS)], B (1833% IS), and D = condensed milk (22.23% IS). Journal of Dairy Science Vol. 77, No.4, 1994
=condensed milk (15.40% IS), C =condensed milk
903
REDUCED FAT CHEDDAR CHEESE
down products at 4, 8, and 12 wk than did TRT A cheeses.
lipolysis. An increase in proteinase addition may promote a faster rate of desired texture development, especially with slower ripening cheeses. Analysis of cheese samples by SDS-PAGE showed definite proteolytic effects of concentration of cheese milk and ripening TRT. Protein groups quantified through the use of densitometry showed differences in proteolytic activity, each greater than the next in the order ENZ, ET, control, and TRT A, B, C, and D (P < .05) (Tables 9, 10, and II). Several important variables influence cheese ripening, including the salt-to-moisture ratio, milk quality, ripening temperature, bacterial count and species involved, residual coagulant in the cheese curd, and the effects of different manufacturing procedures (15, 19). Bacterial growth factors include the salt and moisture content, pH at milling, cooking temperature, and populations of nonstarter lactic acid bacteria, which may be added to increase the ripening rate. Increased proteolytic activity in ENZ and ET cheeses, indicated by increased amounts of
DISCUSSION
All cheeses produced had at least a onethird reduction in fat content (dry weight basis) and :S;125% of the maximum allowable moisture content of the standard cheese variety. In a preliminary experiment, equal amounts of the commercial enzyme preparation were used for preparation of full fat and RFCC and ripened at 6 to 7°C. The full fat Cheddar cheese had excellent flavor at 9 wk, but the RFCC had developed rancidity. At 16 wk, the RFCC was rancid, but the full fat cheese maintained good flavor and texture development. Hargrove et al. (11) suggested that low fat cheeses with added enz.yme produce extensive rancidity. Milk fat may also play the role of masking off-flavor in cheese (22). This role of milk fat may not be fully available in RFCC. Thus, with a reduction in fat content in the cheese milk, the rate of lipase addition would also have to be reduced to prevent excessive
MW 97,400 66,200 45,000 31,000 21,500 14,400
u s2 -CN -----us,-CN ----.:. B-CN--
B-LG _ _ para-K-CN___
u-LA _ _
Control
ET
ENZ
Figure 1. The SDS-PAGE lanes of reduced fat Cheddar cheese from treatment A control uncondensed milk, 12 wk of age. Lane I, low molecular weight (MW) standard containing phosphorylase b (MW 97,400), BSA (MW 66,200), ovalbumin (MW 45,000), bovine carbonic anhydrase (MW 31,(00), soybean trypsin inhibitor (MW 21,500), and egg white lysozyme (MW 14,400). Treatments: control ripening (6 to 7"C), ET = elevated temperature ripening (II "C), ENZ = enzyme ripening (6 to 7'C). Journal of Dairy Science Vol. 77, No.4, 1994
904
BRANDSMA ET AL.
TABLE 8. Acid-soluble nitrogen 1 in reduced fat Cheddar cheese from condensed milk across all ages. Treatment 2
Ripening treatment 3
A
B
C
D
Conlrol ET ENZ
.76a.A .89 a.B .96a.C
.74 ab .A .82 b.B .84b.B
.72 bc •A .82 b.B .84b.B
.69 c.A .78 c.B .79 d.B
a.b.CMeans in rows with like superscripts do not differ (P > .05). A.B.cMeans in columns with like superscripts do not differ (P > .05). !Means across ripening treatments of five replicates; standard error of the mean
= .01.
2Treatments: A = uncondensed milk [10.27% total solids (TS)], B = condensed milk (15.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% TS). 3Control ripening at 6 to 7'C, ET
= elevated temperature
soluble nitrogen, did not improve cheese texture, perhaps because of insufficient proteolysis or because of the nature of proteolytic breakdown products. The retardation of ripening was likely due to compositional imbalances in the condensed milk cheeses, such as higher mineral contents, which decreased bacterial activity (2) as the concentration of cheese milks increased.
ripening (I I 'C), and ENZ
= enzyme
ripening (6 to TC).
Temperature also determines the rate of ripening; the rate of flavor and texture development is influenced by proteolytic and lipolytic activities of bacterial enzymes. The optimal temperature may be higher to allow for maximal proteolytic effect of the enzyme because this effect also occurred in RFCC from UF milk concentrated five times (20). Elevated temperatures to accelerate the rate of
TABLE 9. Specific casein fraction percentages! (0<51-. 0<52-' and i3-caseins) of reduced fat Cheddar cheese from condensed milk. Age of cheese Treatment2
o wk
4 wk
8 wk
12 wk
(%) A Control ET ENZ
47.J&·A 47.J&·A 47. J&.A
36.7 b.BC 35.3 b.C 30.7 b.E
30.3c.CD 25.7 c.G 22.OC· H
27.6 d.CD 24.9c.FG 18.7d.H
B Control ET ENZ
43.9 a.B 43.9 a.B 43.9 a.B
36.1 b.BC 34.9 b.C 32.6 b.D
30.OC· CD 28.6 c.DE 27.9c.EF
28.4c.c 26.Qd·DEF 23.5 d .G
C Control ET ENZ
45.4a.AB 45.4·· AB 45.4··AB
36.2 b.BC 35.0 b.C 32.7 b.D
33.4c.B 29.5 c.CDE 26.2c.FG
32.lc.B 28.OC·c 25.4c.EF
D Control ET ENZ
45.2·· B 45.2··B 45.2·· B
38.5 b.A 37.1 MB 36.4b.BC
35.2c.A 34.1 c.AB 30.6c.C
33.9c.A 32.9c.A 26.8 d.CDE
a.b.c.dMeans in rows with like superscripts do not differ (P > .05). A.B.C.D.E.FMeans in columns with like superscripts within same milk concentration and age do not differ (P > .05). I Mean
of five replicates; standard error of the mean
= .62.
2Treatments: A = uncondensed milk [10.27% total solids (TS)]. B = condensed milk (15.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% TS). Control ripening at 6 to 7'C. ET = elevated temperature ripening (11 T). and ENZ = enzyme ripening (6 to TC). Journal of Dairy Science Vol. 77. No.4. 1994
905
REDUCED FAT CHEDDAR CHEESE
TABLE 10. Specific protein breakdown percentages' (components between l3-casein and para-K-casein in SDS-PAGE) of reduced fat Cheddar cheese from condensed milk. Ripening treatment 2
Age of cheese
o wk
4 wk
8 wk
12 wk
2S.2c.A 27.7 c,B 31.2c,c
28.9C·A 30.8d,B 3S.3d,c
(%) Control ET ENZ
18.4··A 18.4··B 18.4',c
22.2 b,A 23.7 b.B 26.lb.C
•.b.c.dMeans in rows with like superscripts do not differ (P > .OS). A,B.cMeans in columns with like superscripts do not differ (P > .OS). IMeans across all milk concentrations of five replicates; standard error of the mean
= .39.
2Control ripening at 6 to TC, ET = elevated temperature ripening (l1"C), and ENZ = enzyme ripening (6 to TC).
ripening must be used with caution; off-flavor development can occur from uncontrolled proteolysis, The condensed milk cheese generally had a good Cheddar flavor that was stronger than that of the uncondensed milk cheese, but overall flavor scores of the condensed milk control cheeses were lower than those of the uncondensed control. The increased yield and longer shelf-life than most commercial RFCC are advantages of condensed milk (2). Also, less lipolytic activity is needed in RFCC to achieve an acceleration effect of the aroma and taste. Further study of protease addition to RFCC is needed.
The high moisture content in cheese from A ET led to off-flavor development from uncontrolled proteolysis, With ET cheeses from condensed milk, the higher mineral content led to excessive moisture expulsion and a dry, short texture with some off-flavor development in addition to slower ripening. Basic compositional problems of the cheese milk and cheesemaking procedures need to be resolved before elevated temperatures are used to ripen for acceleration of the cheese maturation process, Treatment of RFCC with enzymes is a viable alternative to accelerate the ripening process, but more research is needed to determine
TABLE II. Specific protein breakdown percentages (lower molecular weight than para-K-casein) of reduced fat Cheddar cheese from condensed milk. Age
o wk
4 wk
8 wk
12 wk
(%) Treatments I A B C D Ripening treatment 2 Control ET ENZ
8.6"A 1O.lc.8 IO.S·,8 9.4·· AB
14.4b.A 14.8b,A IS.Ob,A 12.S b,B
17.sc,A 16.7c,A 16.2b.A B.Sb,8
20.2d,A 17.8c.8 18.6c.8 16.OC·c
9.6·,A 9.6"A 9.6·,A
14.0b,A 14.Sb,A 14.0b,A
IS.3 c,A 16.6c,B lS.9c,AB
17.2d,A 19.1d,B 18.1 d,C
•.b.c.dMeans in rows with like superscripts do not differ (P > .OS). A.B,cMeans in columns with like superscripts within concentration or ripening treatment do not differ (P > .OS). IMeans across all ripening treatments of five replicates. Treatments: A = uncondensed milk [10.27% total solids (TS)], B = condensed milk (1S.40% TS), C = condensed milk (18.33% TS), and D = condensed milk (22.23% IS); standard error of the mean = .47. 2Means across all milk concentrations of five replicates. Control ripening at 6 to 7"C, ET = elevated temperature ripening (I I"C), and ENZ = enzyme ripening (6 to TC); standard error = .23. Journal of Dairy Science Vol. 77, No.4, 1994
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BRANDSMA ET AL
the best sources of enzymes and the correct blend of proteases for texture development. CONCLUSIONS
Accelerated ripening is an alternative method for flavor development in RFCC from condensed milk. The RFCC from condensed milk had firm, crumbly body but had a higher Cheddar flavor score than control cheeses. The rate of ripening, as indicated by soluble nitrogen and electrophoresis, was slower in the condensed milk cheeses than in the control cheeses. Rancid flavor developed rapidly in all cheeses made with added enzyme; hence, enzyme preparations with balanced proteases and lipases should be selected. ACKNOWLEDGMENTS
The authors thank the Minnesota-South Dakota Dairy Foods Research Center for funding this project and W. Lee Tucker, South Dakota State University, for providing valuable help with statistical analysis. REFERENCES
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