Primary proteolysis and textural changes during ripening in Cheddar cheeses manufactured to different fat contents

International Dairy Journal 10 (2000) 151}158

Primary proteolysis and textural changes during ripening in Cheddar cheeses manufactured to di!erent fat contents Mark A. Fenelon, Timothy P. Guinee* Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, County Cork, Ireland Received 3 September 1999; accepted 3 April 2000

Abstract The e!ects of varying fat content in Cheddar cheese, from 6.3 to 32.5 g 100 g\, on changes in pH, primary proteolysis and texture were monitored over a 225 d ripening period. Reduction in the fat content resulted in signi"cant (P(0.05) increases in pH, moisture and protein contents and decreases in the concentration of moisture in the non-fat substance. The increase in pH as the fat content increased was attributed to the concomitant decrease in the lactate-to-protein ratio. Polyacrylamide gel electrophoresis showed that the concentration of intact casein decreased in all cheeses during ripening and that the rate of decrease was not a!ected by the fat content. However, for a given concentration of casein, a -casein was degraded more slowly, and b-casein more rapidly, as the fat  content was reduced. The slower degradation of a -casein with decreased fat content coincided with a decrease in the ratio of residual  chymosin activity to protein in the cheese. At most ripening times, reduction in the fat content resulted in signi"cant increases in the concentration of intact casein, fracture stress, fracture strain, and cheese "rmness. The e!ects of fat reduction on proteolysis and rheology are probably due to the interactive e!ects of the concomitant changes in composition.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Proteolysis; Texture; Fat content; Cheddar cheese

1. Introduction Cheese is a nutritious versatile food that contains a high concentration of essential nutrients relative to its energy content (Fox, O'Connor, McSweeney, Guinee & O'Brien, 1996). Yet, rennet curd cheeses do not universally enjoy a healthy image because of the association between dietary fat and possible health-related problems. Consequently, reduction in the fat content of cheese is considered a major focus for consolidating and increasing cheese consumption (Market tracking Int., 1998). However, reduction in fat content of cheese generally leads to impairment of the textural characteristics (Emmons, Kalab, Larmond & Lowrie, 1980; Bryant, Ustunol & Ste!e, 1995). Changes in proteolysis and texture related to maturation of full-fat cheese have been extensively investigated and reviewed (Creamer & Olson, 1982; Visser, 1991;

* Corresponding author. Tel.: #353-25-42204; Fax: #353-2542340. E-mail address: [email protected] (T.P. Guinee).

Prentice, Langley & Marshall, 1993; Fox et al., 1996). Generally, ageing of Cheddar cheese results in a reduction in the levels of intact casein and a concomitant decrease in "rmness, fracture stress, and fracture strain (Creamer & Olson, 1982; Prentice et al., 1993). Comparatively fewer studies have been reported for cheeses of reduced fat content. Moreover, in most of these studies investigations have principally concentrated on the effects of various parameters such as starter culture/starter culture adjuncts (Ardo, Larsson, Mansson & Hedenberg, 1989; Banks, Brechany & Christie, 1989), fat replacers (Fenelon & Guinee, 1997) or cheesemaking conditions (Guinee et al., 1998) on the proteolysis and texture of a reduced-fat cheese of a given fat content. In contrast, few direct comparisons between cheeses of di!erent fat contents have been made. Reduction in fat content of Cheddar cheese, in the range 32.5}6.3 g 100 g\, resulted in lower levels of primary proteolysis, as measured by the levels of pH 4.6 soluble N as % total N (Banks et al., 1989), and in increases in fracture stress, hardness, cohesiveness and springiness (Mackey & Desai, 1995). The change in the rheological characteristics has been attributed in part to the increased concentration of the

0958-6946/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 4 0 - 6

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para-casein matrix per unit cross-sectional area (Mistry & Anderson, 1993; Guinee et al., 1998). At a commercial level there is an increased demand for cheeses of di!erent fat levels to target di!erent consumer segments and applications. The objective of the current study was to investigate the e!ects of incremental fat reduction in the range 32.5}6.3 g 100 g\ on the rheological and proteolytic changes in Cheddar cheese during maturation.

2. Materials and methods 2.1. Manufacture of reduced-fat Cheddar cheese Mid-lactation milk, obtained from the Institute herd, was standardized to give four separate protein-to-fat ratios: 0.95, 1.66, 2.15 or 6.2. The milk batches were held overnight at (103C, pasteurized at 723C;15 s, and pumped into cylindrical jacketed stainless steel 500 L cheese vats, with variable speed cutting and stirring (APV Schweiz AG, CH-3076, Worb 1, Switzerland). The milk was inoculated (1.4 g 100 g\) with a starter comprised of Lactococcus lactis ssp. cremoris AM2 (obtained from the Moorepark stock collection) and Lactococcus lactis ssp. lactis 303 (Chr. Hansen Ireland Limited, Rohan Industrial Estate, Little Island, Co. Cork.) at a ratio of 1.5 : 1. The cultures were grown separately overnight in 10% (wt/wt) reconstituted skim milk powder (10 g 100 mL\) that had been heat treated at 1203C for 30 min. After a 30 min ripening interval, Chymosin (Double Strength Chy-max, 50,000 MCU mL\, P"zer Inc. Milwaukee, WI, USA), diluted in 1 : 30 with deionized water, was added at a level of 2.19 mL kg\ to the milk. The curd was cut, the curd/whey mixture was heated to 383C over an interval of 35 min and the whey was separated at pH 6.15. The curd was cheddared, milled at pH 5.35, salted at a rate of 2.75% and allowed to stand for &20 min before pressing overnight at 264.6 kPa. Subsequently, the cheeses were vacuum wrapped and stored at 43C for 1 month and then at 73C for the remainder of the ripening period. Three replicate trials were carried out during the summer months, i.e., from a spring calving herd. The di!erent products obtained are coded as FFC, RFC, HFC and LFC for full-fat, reduced-fat, half-fat and low-fat Cheddar cheeses, resp. 2.2. Coagulant activity and chemical analysis of cheese Cheeses were sampled at 28}30 d, grated, and analysed for moisture, protein, fat and pH by methods described by Fenelon and Guinee (1999). Lactic acid content was determined using a Boehringer Mannheim test kit, UV method (Cat No. 1112821, Boehringer BCL, Blackrock, Dublin, Ireland).

Residual coagulant (chymosin) activity was measured after 30 and 90 d of maturation by the method of Hurley, O'Driscall, Kelly and McSweeney (1999). The levels of pH 4.6 soluble N (pH 4.6SN) were measured at 1, 30, 60, 90, 120, 180 and 225 d using a modi"cation of the method described by Kuchroo and Fox (1982). A grated sample (&70 g) was homogenized using a stomacher (Stomacher, Lab-Blender 400) for 5 min. The resultant homogenate was placed in a water bath at 503C for 1 h after which it was "ltered through glass wool. The pH of the "ltrate was adjusted to 4.6 using HCl (1 N), followed by centrifugation, at 2460 g;20 min at 43C. Immediately after centrifugation the supernatant was "ltered through Whatman 113V wet strengthened folded "lter paper (Whatman International Ltd., Maidstone England) and the N content determined by macroKjeldahl (IDF, 1993). The level of intact casein (g 100 g\ cheese) in the cheese was calculated using the following formula: Intact casein"6.38;[total N (g 100 g\)-pH 4.6 soluble N (g 100 g\)], where the factor 6.38 was used to convert N to casein. In the above equation it was assumed that all the protein in the cheese is casein; this assumption is reasonable as undenatured whey protein accounts for only &1 g 100 g\ of total protein in the full-fat cheese. 2.3. Urea}polyacrylamide gel electrophoresis Electrophoresis in alkaline polyacrylamide gels (PAGE) was performed on a Protean II xi vertical slab gel unit (Bio-rad Laboratories Ltd., Watford, Herts., UK), using a separating and a stacking gel, according to the method of Andrews (1983). Cheese samples (20 mg) were dissolved in 1 mL of sample bu!er and incubated at 553C for &10 min. The gels (1 mm thick) were pre-run at 280 V for &40 min prior to sample application. Samples from FFC, HFC and LFC cheeses were analysed by PAGE at 1, 60, 120 and 180 d; and those from the RFC at 120 and 180 d. Cheese samples were applied to the gel, via microsyringe, both on a total weight basis and on a protein weight basis. It was envisaged that the former gel would allow quanti"cation of the relative concentrations of the major casein/peptide fragments in the cheeses, while the latter gel would enable a comparison of the extent of protein degradation among the cheeses. Currents of 280 and 300 V were applied as samples passed through the stacking and separating gels, respectively. The gels were stained overnight using the method of Blakesley and Boezi (1977) and de-stained in repeated changes of distilled water. 2.4. Rheological measurements The cheeses were sampled for rheological analysis at 60, 120, 180 and 225 d. Cylindrical samples (r"30 mm;

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Table 1 The e!ect of reducing fat content on the compositions of Cheddar cheese Cheese code


Fat

Moisture

MNFS (g 100 g\)

S/M

Protein

Lactate

Lactate/ protein

pH

FFC RFC HFC LFC SED

32.5a 22.8b 17.5c 6.3d 0.21

37.5a 40.5b 42.3c 45.7d 0.23

55.7a 52.5b 51.3c 48.8d 0.35

4.69a 4.31ab 4.44ab 4.16b 0.19

25.9a 31.3b 33.5c 39.9d 0.29

1.33a 1.36a 1.39a 1.52a 0.12

0.051a 0.044ab 0.041ab 0.038b 0.004

5.29a 5.23ab 5.19b 5.23ab 0.03

Values within a column not sharing a common letter di!er, P(0.05.
FFC"full-fat Cheddar, RFC"reduced-fat Cheddar, HFC"half-fat Cheddar, LFC"low-fat Cheddar. MNFS"moisture in the non-fat substance. S/M"salt-in-moisture. Measured at 90 d. Standard error of di!erence; degrees of freedom"6.

h"29 mm) were cut from cheeses immediately after removal from refrigerated storage (73C), placed in a sealed plastic bag and equilibrated at 83C overnight. Cooled (83C) samples were removed from refrigerated storage and immediately compressed to 30% of their original height at a rate of 50 mm min\ on a Universal Testing Machine (Model 4301; Instron Ltd., High Wycombe HP12 35 Y, UK) at room temperature. For each cheese at any given sampling time, there were "ve replicate samples. The force required to compress the cheeses to 30% of their original height was taken as the "rmness value. 2.5. Statistical analysis A randomized complete block design which incorporated the four treatments (FFC, RFC, HFC and LFC), and three blocks (replicate trials) was used for analysis of the response variables relating to cheese composition and residual chymosin activity (Tables 1 and 2). Analysis of variance (ANOVA) was carried out using an SAS (1995) procedure where the e!ects of treatment and replicates were estimated for response variables. Duncan's multiple-comparison test was used as a guide for pair comparisons of the treatment means. The level of signi"cance was determined at P(0.05. The experimental design for the response variables pH, intact casein, "rmness, fracture stress and fracture strain was a split plot with three replicates. The main plot factor was fat level, i.e., 32.5, 22.8, 17.5 and 6.3 g 100 g\, and the sub-plot factor was ripening time. Analysis of variance was carried out using a general linear model (GLM) procedure of SAS (1995) where the e!ects of treatment and replicates were estimated. Fisher's di!erence test was used to determine whether statistically signi"cant di!erences occurred among means. All di!erences considered as signi"cant are at least P(0.05. Urea-polyacrylamide gel electrophoresis (urea}PAGE) was performed on the cheeses from each trial at di!erent sampling times. The results from urea}PAGE are pre-

Table 2 Residual chymosin activity (RU, rennet units) in Cheddar cheeses of di!erent fat contents at 30 or 90 d Cheese code


FFC RFC HFC LFC SED

RU kg\ cheese

RU kg\ protein

30 d

90 d

30 d

90 d

13.64b 16.35ab 16.93ab 18.49a 1.57

13.99c 14.89bc 15.77b 18.26a 0.55

0.52a 0.52a 0.50a 0.48a 0.047

0.54a 0.48b 0.47b 0.47b 0.01

Values within a column not sharing a common letter di!er, P(0.05.
FFC"full-fat Cheddar, RFC"reduced-fat Cheddar, HFC"halffat Cheddar, LFC"low-fat Cheddar. SED"standard error of di!erence; degrees of freedom"6.

sented as observations and supportive data but were not statistically analysed.

3. Results and discussion 3.1. Cheese composition The gross compositions of the cheeses are summarized in Table 1. Reduction in fat content resulted in signi"cant increases in the contents of moisture and protein, and reductions in the levels of salt-in-moisture and moisture in the non-fat substance (MNFS). The reduction in the salt-in-moisture level with decreasing fat content was due to the concomitant increase in cheese moisture; there was no di!erence in the salt level, on a total weight basis, among any of the cheeses. 3.2. Age-related changes in pH The pH of all cheeses except FFC increased with ripening time, with the pH at 180 d ranging from &5.5

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Fig. 1. The e!ect of fat content on pH over a 225 d ripening period in: full-fat, FFC (䊐); reduced-fat, RFC (䊏); half-fat, HFC (*); and low-fat, LFC (䢇) Cheddar cheese. Values presented are the means from 3 replicate trials.

relationship between pH and lactate level in full-fat Cheddar, in which the lactate-to-protein ratio was varied by curd washing and/or addition of lactose to the milk or to the whey immediately after cutting (Hu!man & Kristo!ersen, 1984; Fox & Wallace, 1997). For a given level of cheese non-fat substance, which consists mainly of casein and ash, a reduction in the moisture content, and hence, in the levels of lactose and lactic acid, is expected to result in a greater increase in pH for a given level of proteolysis and production of basic compounds such as amino acids, amines and/or ammonia (Bouzas, Kantt, Bodyfelt & Torres, 1993; Lucey & Fox, 1993; Mistry & Kasperson, 1998; Fenelon et al., 1999). There was an inverse relationship between cheese pH and MNFS content at all ripening times '40 d (Fig. 2). Other factors e.g., di!erences in the level of ammonia production may have also contributed to the increase in pH as the fat content was reduced. There was a signi"cant (P(0.05) decrease in the pH of the full-fat cheese between 1 and 120 d, an occurrence that has been observed also by others (Hu!man & Kristo!ersen, 1984; Bouzas et al., 1993). This trend may re#ect the e!ect of the conversion of residual lactose to lactic acid in the cheese. The concentrations of protein and hence phosphate (Fenelon & Guinee, 1999), which are the major bu!ering substances in cheese (Lucey & Fox, 1993), were the lowest in the FFC cheese. 3.3. Changes in pH 4.6 soluble N The pH 4.6SN, as a percentage of total N, increased in all cheeses during maturation (Fig. 3). There was a signi"cant e!ect of the interaction between time and fat level on the concentration of pH 4.6SN (Table 3), with the value for the FFC generally becoming greater than that of the LFC as ripening time advanced. The lower levels of pH

Fig. 2. Relationship between moisture in the non-fat substance and pH of Cheddar cheeses at 120 d (䊐) and 225 d (䊏) of ripening. Values presented are the means from 3 replicate trials.

in the LFC to &5.2 in the FFC (Fig. 1). There was a signi"cant e!ect of the interaction between ripening time and fat content on the pH, as indicated by the divergence in the pH/time curves (Fig. 1; Table 3). The increase in cheese pH as the fat content was reduced may be attributed to the concomitant decrease in the MNFS content and, hence, lactate-to-protein ratio. The levels of lactate tended to increase with decreasing fat level, while the lactate-to-protein ratio decreased signi"cantly on reducing the fat level from 32.5 to 6.3 g 100 g\ (Table 1). The relationship between cheese pH and fat content in Cheddar is analogous to the

Fig. 3. Changes in pH 4.6 soluble N expressed as g 100 g\ of cheese N in: full-fat, (FFC), reduced-fat (RFC), half-fat (HFC) and low-fat (LFC) Cheddar cheeses during ripening. Values presented are the means from 3 replicate trials.

13

467

0.001 0.19 9849 1175 3 9

4453 13 0.54 48 2.4 48 0.002 48

Cheeses were sampled for rheological analysis at 60, 120, 180 and 225 d. However, at 60 d the LFC cheese failed to fracture at 70% sample compression; hence, the data for fracture stress and strain at 60 d were omitted from the statistical model.

13

17.2

0.01 0.94 155 4.2 2 5 0.05 0.49 17,793 4162 0.001 0.99 64.3 0.19 0.05 0.01 6 18

0.001 0.001

6 18

561 7.9

0.001 0.001

6 18

2 5

0.001 359,473 1818 3 6 0.05 280 33.7 3 6 0.001 325,850 3937 0.001 730 0.10 0.008

Main plot Fat content Error Sub-plot Time Interaction (fat;time) Error

3 6

0.001

3 6

114 2.17

0.001

3 6

3 6

MS df MS df P MS df

MS

P

df

MS

P

df

df

MS

P

Fracture strain Fracture stress Intact casein pH 4.6SN pH Factors

Table 3 Mean squares (MS) and probabilities (P) for age-related changes in pH, pH 4.6 soluble N (pH 4.6SN), intact casein, fracture stress, fracture strain and "rmness

P

Firmness

P

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4.6SN as fat content was reduced (Fig. 3) may be due in part to the concomitant decreases in the level of MNFS (Table 1) (Creamer, 1971; Pearce & Gilles, 1979) and the chymosin-activity-to-protein ratio (Table 2). Lawrence and Gilles (1980) concluded that small increases in MNFS (e.g., 2}4 g 100 g\) lead to a relatively large increase in the availability of water, and, in turn to increases in the activity of microorganisms and enzymes and the degree of proteolysis. The level of pH 4.6SN, expressed as g 100 g\ cheese, increased in all cheeses during maturation, but was not signi"cantly a!ected by fat content (results not shown). This trend suggests that the reduction in pH 4.6SN, expressed as g 100 g\ total N, as the fat content decreased was compensated for by the concomitant increase in protein content. Hence, fat content had no signi"cant e!ect on the concentrations of pH 4.6SN when expressed as g 100 g\ cheese. Casein degradation during maturation was paralleled by a simultaneous reduction in the levels of intact casein, the levels of which at all ripening times were in the order LFC'HFC'RFC'FFC. The rate of decrease in the levels of intact casein per 100 g cheese with ripening time (&0.022 g 100 g\ cheese d\) was not signi"cantly in#uenced by fat content (Fig. 4). The results indicate that higher fat content in the cheese was accompanied by a decrease in the content of intact casein (g 100 g\ cheese) and an increase in the rate of protein degradation (i.e., g protein broken down 100 g\ protein present). 3.4. Urea}polyacrylamide gel electrophoresis (urea}PAGE) urea}PAGE gel electrophoretograms of the di!erent cheeses applied to the gel on the basis of a "xed weight of cheese, or a "xed weight of protein, are shown in Figs. 5a and b, respectively. While reduction in fat content had little e!ect on the degradation pattern, it resulted in di!erences in the relative concentrations of the various casein fractions. When loaded on a total weight basis, the levels of intact a -casein and b-CN increased as the fat level  decreased, a trend expected because of the inverse relationship between the fat and protein levels in the cheese. At all ripening times, reduction in fat content was paralleled by increases in the intensities of c-caseins, b-casein f (29}209), b-casein f (106}209), and b-casein f (108}209). At ripening times '60 d, the concentration of the a  casein f (24}199), and thus the complementary Nterminal fragment a -casein f (1}23), increased as the fat  content was reduced. In contrast, the breakdown product of a -casein f (24}199), i.e., a -casein f (102}199) (Fox   et al., 1996; McSweeney & Fox, 1997), was scarcely in#uenced by the fat content of the cheese. In the maturing cheese, a -casein f (24}199) and a -casein f (1}23) are   hydrolysed into smaller peptides by the chymosin and

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Fig. 4. Changes in the estimated levels of intact protein expressed as g 100 g\ cheese (b) in: full-fat, FFC (䊐); reduced-fat, RFC (䊏); half-fat, HFC (*); and low-fat, LFC (䢇) Cheddar cheeses during ripening. Intact casein was estimated from data on total protein and pH 4.6 soluble N, as described in text.

the starter cell wall proteinase, respectively (Fox et al., 1996). These peptides are further degraded to smaller peptides and amino acids by starter peptidases (Van den Berg & Exterkate, 1993). Hence, a reduction in the fat content of Cheddar cheese should result in higher levels of free amino acids present in the cheese. When the gels were loaded with a "xed protein level (Fig. 5b), the di!erences in the concentrations of intact a - and b-caseins between the cheeses of di!erent fat  content were less than those when the gels were loaded on a total weight basis (Fig. 5a). However, reduction in fat level resulted in greater degradation of b-casein and accumulation of c-caseins, especially at ripening times *120 d. The concentrations of intact b-casein in the FFC at ripening times 120 and 180 d were signi"cantly higher than those in either the HFC or LFC which did not signi"cantly di!er. Densitometric analysis of the electrophoretograms showed that the concentrations of intact b-casein in the FFC and LFC cheeses at 180 d amounted to &90 and 58% of those present at 1 d, respectively. The increase in the concentration of c-caseins as the fat content was reduced was probably associated with the concomitant increase in cheese pH, which is expected to enhance the activity of the native milk alkaline proteinase plasmin (Richardson & Pearse, 1981; Gru!erty & Fox, 1988). Plasmin hydrolyses all caseins, especially a - and b-caseins, with the hydrolysis  f the latter resulting in the formation of c -, c - and   c -caseins (Fox et al., 1996). The increase in pH as the fat  content decreased may also a!ect the degree of hydration and aggregation of the b-casein (Creamer, 1985) which in turn may a!ect its susceptibility to hydrolysis by chymosin and plasmin. Moreover, the slight reduction in

Fig. 5. Urea-polyacrylamide gel electrophoretograms of sodium caseinate (lane 1) and cheeses (lanes 2}15), loaded with a "xed weight of cheese (Gel a) and "xed weight of N (Gel b). The gels were loaded as follows: full-fat (FFC) at 1, 60, 120 and 180 d (lanes 2, 5, 8 and 12, respectively), reduced-fat (RFC) at 120 and 180 d (lanes 9 and 13), half-fat (HFC) at 1, 60, 120 and 180 d (lanes 3, 6, 10 and 14) and low-fat (LFC) at 1, 60, 120 and 180 d (lanes 4, 7, 11 and 15).

the salt-in-moisture concentration as the fat level was decreased may also have contributed to the increased breakdown of b-CN in the low-fat cheese, due to an enhanced activity of residual chymosin (Fox & Walley, 1971; Kelly, Fox & McSweeney, 1996). In contrast to the trend noted for b-casein breakdown, the degradation of a -casein decreased as the  fat content was reduced (Fig. 5b). This trend was probably due to the decrease in residual rennet activity (RU kg\ protein) as the fat content was lowered (Table 2). Reduction in the fat level had little e!ect on the concentration of a -casein f (24}199) but resulted in  lower levels of its degradation product a -CN  f (102}199) at ripening times *60 d. The latter e!ect may again be due to the lower chymosin activity relative to the casein content and lower concentration of MNFS (Table 1). In addition, the higher pH of the lower fat cheeses was less favourable to the proteolytic activity of residual rennet (Tam & Whitaker, 1972; Vanderpoorten & Weckx, 1972). 3.5. Rheology The fracture stress, fracture strain and "rmness of all cheeses decreased signi"cantly with ripening time '2 months (Figs. 6a}c; Table 3). These decreases were observed also by others (Creamer & Olson, 1982; Visser,

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the fracture stress and "rmness of the LFC were about three- and two-fold higher than those of FFC cheese, respectively. Moreover, at ripening times (120 d, the LFC failed to fracture when compressed to 30% of its original height, indicating that the fracture stress was greater than 1400 kPa. This increase in "rmness, also observed by others (Bryant et al., 1995; Mackey & Desai, 1995), may be attributed at least in part to the concomitant increase in the level of intact casein per unit weight of cheese (Fig. 4) and the reduction in fat level (Prentice et al., 1993). Other factor that may contribute to the change in rheology with fat content is the degree of hydration or aggregation of the casein as a!ected by pH and calcium content (Creamer, 1985; Sood, Gaind & Dewan, 1979).

4. Conclusions Reduction in the fat content of Cheddar cheeses, in the range 32.5}6.3 g 100 g\, resulted in increases in the moisture and protein contents and pH, and decreases in the MNFS content and in the lactate-to-protein and chymosin-activity-to-protein ratios. Primary proteolysis as measured by pH 4.6 soluble N, expressed as g 100 g\ total N, decreased with decreasing fat content in the cheese. However, primary proteolysis as measured by the level of pH 4.6SN, expressed as g 100 g\ cheese, was not signi"cantly in#uenced by fat content. The concentrations of intact a - and b-caseins per unit weight of cheese  increased markedly as the fat content decreased. However, the rate of a -casein degradation was greater in the  higher fat cheeses while that of the b-casein casein was higher in the low-fat cheeses. The increase in fracture stress, fracture strain and "rmness as the fat content was reduced probably re#ects the concomitant changes in composition (e.g., increase in intact casein content) and degree/type of proteolysis.

Acknowledgements Fig. 6. Changes in (a) fracture stress, (b) fracture strain and (c) "rmness in Cheddar cheeses of di!erent fat content: full-fat (FFC) (䊐); reducedfat (RFC) (䊏); half-fat (HFC) (*); low-fat (LFC) (䢇). Values presented are the means from 3 replicate trials. Broken line indicates that the sample did not fracture on compression.

1991) and may be attributed to the reduction in the levels of intact casein as re#ected by changes in N solubility and urea}PAGE polyacrylamide gel electrophoresis (de Jong, 1978; Creamer & Olson, 1982; Prentice et al., 1993). Reduction in the fat content, and the concomitant increase in the content of intact casein, of the cheese resulted in signi"cant increases in all three rheological parameters. At the end of the 225 d maturation period,

This project was part-funded by the European Union Structural Funds (European Regional Development Fund).

References Ardo, Y., Larsson, P. O., Mansson, L., & Hedenberg, A. (1989). Studies of peptidolysis during early maturation and its in#uence on low-fat cheese quality. Milchwissenschaft, 44, 485}488. Andrews, A. T. (1983). Proteinases in normal bovine milk and their action on caseins. Journal of Dairy Research, 50, 45}55. Banks, J. M., Brechany, E. Y., & Christie, W. W. (1989). The production of low fat Cheddar cheese. Journal of Society of Dairy Technology, 42, 6}9.

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