International Dairy Journal 30 (2013) 53e58

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International Dairy Journal

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Effects of variation in cheese composition and maturation on water

activity in Cheddar cheese during ripening
D.K. Hickey a, *, T.P. Guinee b, J. Hou b, M.G. Wilkinson a
a
Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland
b
Teagasc, Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Changes in the water activity (aw) of experimental Cheddar cheeses with different compositions were
Received 18 July 2012 measured over a 270 d ripening period. The ranges of key compositional parameters were typical of
Received in revised form those found in commercial Cheddar cheese: moisture (35.6e38.6%), fat (29.9e33.4%), salt (1.4e2.0%),
18 November 2012
salt-in-moisture (3.8e5.2%), moisture-in-non-fat substances (MNFS, 52.4e56.3%) and pH (5.0e5.7).
Accepted 20 November 2012
Linear regression analysis indicated that at ripening times
30 d, the aw was inversely correlated with
salt-in-moisture, while at 270 d it was positively correlated with levels of moisture and MNFS, but
negatively with fat content. The aw decreased significantly (P < 0.001) during ripening, from a mean of
w0.965 at 1 d to w0.956 at 270 d. The preponderant factor responsible for the decrease in aw was the
increase in proteolysis, as measured using pH 4.6 soluble N and free amino acids.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction cheese varieties (rennet-curd and fresh cheeses) with moisture

Water in cheese is present in ‘bound’ or ‘free’ form, where bound
water is the fraction used to hydrate hydrophilic molecules and
dissolve solutes and is unavailable for biological functions (Ray,
2005). Water activity (aw) is an index of the free water that is
available to contribute to water vapour pressure (Mclaughlin &
Magee, 1998). Fresh cheese curd can be considered as a para-
casein matrix in which the moisture is entrapped (Geurts, Walstra,
& Mulder, 1974). Although water entrapped in cheese pores retains
its solvent capacity, it has a lower aw compared with that of pure
water, because of the presence of NaCl, lactose, and other low mo-
lecular weight solutes (López, Marcos, & Esteban, 1990).
The effects of various factors on the aw of cheese have been
investigated. Marcos, Alcala, Leon, Fernández-Salguero, and Esteban
(1981) determined the relationship between aw and the chemical
composition of twenty six different European cheese varieties with
aw values ranging from 0.91 to 0.99. Water activity was positively
correlated with moisture content (range 32e55 g 100 g1 cheese)
and negatively with the concentration of salt in moisture (range
1.45e11.41 g 100 g1 cheese) and with non-protein nitrogen (0.47e
3.07 g 100 g1 cheese). Owing to the strong relationship between
salt-in-moisture content for retail samples of a range of different
* Corresponding author. Tel.: þ353 (0)25 42470.
E-mail address: dara.hickey@teagasc.ie (D.K. Hickey).
contents >40%, Marcos et al. (1981), Marcos and Esteban (1982) and
Marcos, Esteban, Alcala, and Millan (1983a) concluded that the aw for
these cheeses could be predicted according to the following rela-
tionship: aw ¼ 1  0.033 M, where M is the molarity of sodium
chloride in the moisture phases. For rennet-curd cheeses with
moisture contents <40%, a weaker relationship between aw and
sodium chloride molarity was obtained, due to the contribution of
additional factors such as water-soluble peptides and salts in
reducing the aw. Ruegg and Blanc (1981) similarly reported signifi-
cant inter-variety variation in mean aw (e.g., from a mean of w0.91 in
Parmesan to w0.99 in Cottage cheese), and also noted significant
zonal variation in aw with different brine-salted cheese varieties (e.g.,
from w0.85 to 0.95 in Sbrinz cheese). Water activity was positively
correlated with water content and negatively with concentrations of
nitrogen, ash and salt in the moisture phase; hence, aw was found to
decrease significantly during the ripening of Emmental and Gruyère
cheeses, owing to an increase in water soluble N products. These
trends were confirmed by Saurel, Pajonk, and Andrieu (2004) who
reported zonal variations in the aw of Emmental cheese were
strongly dependent on local salt concentration distributions and on
levels of free amino acids (FAAs).
Bacteria lose water in low aw environments, predisposing them to
plasmolysis and cell lysis (Rahman, 2009). It is well known that lactic
acid bacteria undergo cellular disintegration during manufacture
and ripening of Cheddar cheese, and that this results in cell lysis and
release of intracellular enzymes into the cheese matrix, which, in

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54 D.K. Hickey et al. / International Dairy Journal 30 (2013) 53e58
turn, affects the rate of flavour development (Sheehan, O’Cuinn,
FitzGerald, & Wilkinson, 2009). Reported values of aw in Cheddar
cheese (0.95e0.96; Marcos et al., 1981; Ruegg & Blanc, 1981) suggest
that they may be lower than the limit required for survival by starter
culture lactococci (Beresford, Fitzsimons, Brennan, & Cogan, 2001;
Liu, Asmundson, Gopal, Holland, & Crow, 1998; Ruegg & Blanc, 1981)
and for activity of ripening enzymes (Gobbetti et al., 1999; Liu et al.,
1998; Reid & Coolbear, 1999). Therefore, aw may influence the
microbial evolution and release of starter intracellular enzymes, their
activity and consequently cheese flavour development. However,
information on the aw of Cheddar cheese is very limited, and the
authors are unaware of any studies on the changes during ripening.
Hence, the objective of the current study was to evaluate the effects
of variation in key compositional parameters and ripening on the aw
of experimental Cheddar cheeses.
2. Materials and methods
2.1. Cheese and sampling protocols
A total of 32 Cheddar cheeses were manufactured at pilot scale
(w450 L milk per treatment) in Moorepark Technology Limited in
the period April 2009eMay 2010. The compositions of the cheeses,
coded numerically 1e32, were varied by altering milk protein level
(3.3 or 4.4%, w/w) using ultrafiltration, degree of curd washing in
the cheese vat (equivalent to 0e33 L water per 100 L cheese milk;
Hou, Hannon, McSweeney, Beresford, & Guinee, 2012), and type of
starter culture (different strains of Lactococcus lactis subsp. cremoris
and subsp. lactis). After manufacture, individual cheeses were
vacuum wrapped in Sealed Air CryovacÒ bags (Sealed Air, St. Neots
and Poole, UK), stored at 4  C for two weeks, and subsequently
ripened at 8  C for up to 270 d.
2.2. Cheese composition
Grated cheeses were analysed in triplicate for protein, fat, salt
and moisture using standard IDF methods, as described by Guinee,
Mulholland, Kelly, and Callaghan (2007); the pH was measured on
a cheese slurry according to the British Standards Institution
method (British Standards Institute, 1976). The concentrations of
lactose, galactose and D()- and L(þ)-lactate were determined in
triplicate as described by Hou et al. (2012).
2.3. Water activity determination
The 32 cheeses were analysed for aw at different ripening times:
at 1, 30, 90, 180 and 270 d. Grated cheese (1.5 g) was placed in
a disposable Aqualab water activity sample cup (Decagon Devices,
Inc., Pullman, WA, USA), sealed immediately with the cup lid and
stored at 4  C for no longer than 24 h. The aw was then measured in
triplicate at 21  C using an Aqualab Series 3TE dewpoint electronic
water activity meter with an accuracy of 0.003.
2.4. Aminopeptidase activity
Pep X activity was monitored in triplicate cheese extracts by the
release of AMC (7-amino-4-methylcoumarin) from Gly-Pro-AMC
(Bachem Feinchemikalien, Bubendorf, Switzerland) by the
method of Kilcawley, Wilkinson, and Fox (2002). Cheese extracts
were prepared as follows: 20 g of grated cheese was mixed with
40 mL of 0.05 M potassium phosphate buffer, pH 7.0, stomached for
5 min; 1 mL of homogenate was centrifuged at 13,000  g for 5 min
and enzymatic activity of released intracellular enzymes measured
in the final supernatant. A 50 mL sample of cheese extract in trip-
licate was incubated with 450 mL of 0.111 mM Gly-Pro-AMC in
500 mL of 50 mM TriseHCl, pH 7.0, at 37  C. The reaction was ter-
minated after 15 min by addition of 1 mL of 1.5 M acetic acid. Release
of AMC from the synthetic substrates was determined spectro-
fluorimetrically using a Synergy HT Fluorescent Microplate reader
(Bio-Tek, Winooski, VT, USA) using an excitation wavelength of
360  40 nm and an emission wavelength of 460  40 nm. A
standard curve of AMC fluorescence in the range 0e2 mM was used
to determine the concentration of AMC released (average fluo-
rescence of three samples) by the action of the enzyme. Activity
was expressed as mM of AMC released min1 mL1 of extract.
2.5. Assessment of proteolysis
The percentage of total N soluble at pH 4.6 and individual FAAs
were determined in triplicate as described by Hickey, Kilcawley,
Beresford, Sheehan, and Wilkinson (2006).
2.6. Statistical data analysis
One way analysis of variance (ANOVA) was used to determine
the effect of ripening time on the response variables of the 32
cheeses measured at regular intervals during ripening i.e., aw, pH,
lactose, galactose, D()-, L(þ)-lactate, pH 4.6 soluble N and FAAs
using R (R Development Core Team, 2011) statistical software.
The data for each of the response variables were analysed by
linear regression to establish possible correlations between aw and
the response variables (e.g., pH, salt, fat, salt-in-moisture). The
significance of correlations was calculated using Microsoft Excel
2007 software with n  2$df where n is the actual number of data
points, and df is the degrees of freedom.
3. Results and discussion
3.1. Composition, residual sugar, lactic acid and pH
The compositions of the cheeses at day 30 are shown in Table 1.
In general, the ranges for the different compositional parameters in
this study were similar to those found in commercial Cheddar
cheeses by Guinee, Harrington, Corcoran, Mulholland, and Mullins
(2000) and Guinee, Kilcawley, and Beresford (2008).
The mean level of residual lactose decreased significantly
(P < 0.001) with ripening time but varied considerably within the set
of cheeses at individual ripening times, as reflected by the large
range of values observed especially at times,
90 d (Fig. 1, Table 2).
Such variation may be due to differences in starter cultures and
degree of curd washing (Hou et al., 2012) used in manufacture, and in
the salt content of the cheeses (Turner & Thomas, 1980). Similarly,
large variation in the content of total lactate, which increased sig-
nificantly (P < 0.001) with ripening time and the lactate-to-protein
ratio, was evident (Fig. 1, Table 2), as indicated by the relatively
Table 1
Composition of experimental Cheddar cheeses.a
Parameter Range Mean
Moisture 35.6e38.6 37.5 (2.1)
Fat 29.9e33.4 31.2 (2.4)
Protein 22.7e28.5 25.8 (3.6)
Salt 1.40e1.99 1.79 (7.9)
S/M 3.80e5.24 4.77 (7.5)
MNFS 52.4e55.6 54.5 (1.3)
FDM 48.1e52.1 50.0 (1.5)
a
The experimental set of 32 cheeses were analysed for composition at 30 d.
Values are given as percentages. Values in parentheses are the coefficient of varia-
tion of the mean (%). Abbreviations: S/M, salt in moisture; MNFS, moisture in non fat
substance; FDM, fat in dry matter.
 D.K. Hickey et al. / International Dairy Journal 30 (2013) 53e58 55

Cheddar cheese manufacture (Michel & Martley, 2001) can lead to

significant levels of residual galactose.
The pH varied at individual ripening times, with the range of
spread of values at individual ripening times increasing with rip-
ening time duration (Table 3). This was associated with variation in
the ratio of lactate-to-protein in the cheese, which decreased with
the extent of curd washing (Hou et al., 2012). Several studies have
reported an inverse relationship between Cheddar cheese pH and
lactate content (Guinee et al., 2008; Hou et al., 2012; Thomas &
Pearce, 1981).

3.2. Proteolysis

Changes in the level of pH 4.6-soluble N is routinely used as an

Fig. 1. Concentrations of lactose and total lactate in experimental Cheddar cheeses at
different ripening times; presented data shows the values for 20 cheeses at 1 d and 32
cheeses at all other ripening times.
index of the degree of primary proteolysis in cheese. The pH 4.6-
soluble fraction in Cheddar cheese is comprised mainly of pep-
tides with molecular masses ranging in molecular mass from
>10 kDa to 0.5 kDa, the proportions of which change with ripening
time (Fenelon, Guinee, Delahunty, Murray, & Crowe, 2000). Its
formation is attributed primarily to residual chymosin, with a mi-
nor input from plasmin (Farkye & Fox, 1992) and starter culture
(Fox, 1989). The mean level of pH 4.6 soluble N increased sig-
nificantly (P < 0.001) with ripening time (Table 3). As for lactose,
the degree of variation between individual cheeses decreased with
ripening time, as indicated by the decrease in the CV of the mean
from w24% at 1 d to 8.7% at 270 d (Table 3). A similar trend was
observed for concentration of total FAA, the formation of which is
primarily attributed to the action of starter culture peptidases on
oligopeptides (McSweeney, 2004).
3.3. Enzymatic activity during ripening

large coefficient of variance (CV; w15e21%) observed over the rip-
ening period. This reflects seasonal variation in the composition
(lactose-in-moisture) of the milks used in cheese manufacture
(range of lactose in raw milk was between 4.48% and 4.88%), and the
degree of curd washing (Hou et al., 2012). Hence, washing resulted in
the dilution of the whey surrounding the curd particles (during the
cooking/stirring stage of manufacture) and in effect dilutes the lactic
acid content inside the curd particles, and hence, of the curd as
a whole (Hou et al., 2012; Shakeel-Ur-Rehman, Waldron, & Fox,
2004). The total lactate content in standard, non-washed, full-fat
Cheddar cheese is typically reported to be w1.2% (Guinee et al.,
2008; Jordan & Cogan, 1993). Overall, the mean level of L()-lac-
tate decreased significantly (P < 0.001) during ripening with a con-
comitant significant (P < 0.001) increase in the mean levels of
D()-lactate (Table 2). In agreement with results of previous studies
(Hou et al., 2012; Turner & Thomas, 1980), the levels of residual
galactose were very low (<0.05%, w/w) in all cheeses. Such a trend is
expected as Cheddar cheese is generally made with mesophilic lac-
tococci (e.g., L. lactis subsp. cremoris/lactis), which readily ferment
galactose (Michel & Martley, 2001). However, the recent use of
Streptococcus thermophilus as a component of the starter culture for
Variation in Pep X activity between cheeses was evident at all
ripening times (Table 3), reflecting differences in composition pa-
rameters (e.g., pH, S/M) which affect starter culture cell lysis and
peptidase activities (Gobbetti et al., 1999; Reid & Coolbear, 1999;
Wilkinson, Guinee, & Fox, 1994a; Wilkinson, Guinee, O’Callaghan, &
Fox, 1994b). However, in concurrence with previous studies on
Cheddar cheese (O’Donovan, Wilkinson, Guinee, & Fox,1996; Sheehan
et al., 2009), Pep X activity did not significantly increase with ripening
time. The values at all ripening times (
4.4 mM AMC min1 mL1) were
lower than those (w5e35 mM AMC min1 mL1) reported by Sheehan
et al. (2009), which probably reflects differences in starter culture
types, their initial populations, rates of autolysis, and degrees of per-
meabilisation (O’Donovan et al., 1996; Sheehan, O’Loughlin, O’Cuinn,
FitzGerald, & Wilkinson, 2005).
3.4. Water activity
The mean aw of all cheeses decreased significantly (P < 0.001)
during ripening from 0.964 at 1 d to 0.956 at 270 d (Fig. 2). The aw
values (0.971e0.951) observed during ripening were similar in
magnitude to those reported previously for retail samples of Cheddar

Table 2
Mean, and range of, lactose, lactates and lactate-to-protein ratio in experimental Cheddar cheeses at different ripening times.a

Ripening time (d) n Cheese codes Lactose (%) L(þ)-lactate (%) D()-lactate (%) Total lactate (%) Lactate:protein ratio (%)

Range Mean Range Mean Range Mean Range Mean Range Mean
1 20 13e32 0.007e0.601 0.297 (61) 0.808e1.377 1.023 (17) 0.000e0.011 0.001 (254) 0.809e1.377 1.025 (17) 0.031e0.054 0.040 (18)
30 32 1e32 0.001e0.524 0.171 (78) 0.692e1.516 1.022 (17) 0.000e0.093 0.018 (129) 0.752e1.517 1.040 (17) 0.031e0.060 0.040 (17)
90 32 1e32 0.001e0.544 0.141 (91) 0.581e1.327 0.904 (19) 0.016e0.533 0.142 (76) 0.747e1.372 1.046 (16) 0.029e0.056 0.041 (16)
180 32 1e32 0.000e0.322 0.101 (96) 0.593e1.249 0.868 (21) 0.090e0.849 0.372 (38) 0.853e1.625 1.239 (16) 0.034e0.062 0.048 (15)
270 32 1e32 0.000e0.327 0.068 (123) 0.277e1.307 0.766 (26) 0.184e0.869 0.431 (34) 0.461e1.707 1.187 (21) 0.018e0.068 0.046 (21)
a
Values are percentages; values in parentheses are the coefficient of variation of the mean (%). Abbreviations: n, number of cheeses analysed at the different ripening times.
56 D.K. Hickey et al. / International Dairy Journal 30 (2013) 53e58

Table 3
Mean, and range of, concentrations of pH, pH 4.6 soluble N, total free amino acids and post-proline dipeptidyl aminopeptidase activity at different ripening times.a

Ripening time (d) n Cheese code pH pH 4.6 soluble N (% TN) Total FAAs (mg 100 g1 cheese) n Cheese code Pep X (mM AMC min1 mL1)

Range Meanb Range Meanc Range Meanc Range Meanc

1 20 13e32 5.23e5.39 5.31  0.07 2.3e5.8 3.4 (24) 58.4e280 91.0 (60) 20 13e32 0.3e2.9 1.7 (50)
30 32 1e32 5.28e5.46 5.33  0.09 4.3e15 7.2 (24) 62.3e263 173 (28) 20 13e32 0.5e3.3 1.3 (55)
90 32 1e32 5.30e5.61 5.36  0.11 8.6e17 13 (14) 353e576 443 (13) 20 13e32 0.7e3.3 1.6 (52)
180 32 1e32 5.27e5.63 5.37  0.14 14e24 19 (11) 573e939 737 (15) 20 13e32 0.9e2.9 2.0 (31)
270 32 1e32 5.14e5.58 5.37  0.15 19e29 24 (8.7) 840e1478 1075 (15) 20 13e32 0.9e4.4 2.3 (43)
a
Abbreviations: n, number of cheeses analysed; TN, total N; FAAs, free amino acids; Pep X, post-proline dipeptidyl aminopeptidase.
b
Values  standard deviation.
c
Values in brackets are the coefficient of variation of the mean (%).

cheese, e.g., 0.950e0.960 (Marcos et al., 1981; Ruegg & Blanc, 1981).
Comparison of the current data with those estimated from Ruegg
and Blanc (1981) indicates that the mean aw of the 1 d-old Ched-
dar cheese (w0.964) was lower than that of Emmental and Gruyère
cheeses (w0.982) despite the moisture content of the latter cheeses
(w33e37%, w/w) being generally lower than that of Cheddar (w36e
38%, w/w). This may reflect the lower moisture-to-protein ratios
(and hence lower level of water-soluble solutes: Kosikowski &
Mistry, 1997; Posati & Orr, 1976), lower S/M content, and the
higher scald temperatures in Emmental and Gruyère compared with
Cheddar, which would have a higher degree of protein aggregation
and lower degree of watereprotein interaction. The mean aw values
(w0.955e0.960) in the 270 d-old Cheddar cheese was lower than
those reported by Ruegg and Blanc (1981) for 150 d-old Emmental
(0.97) but higher than that for 150 d-old Gruyère (w0.94) (Ruegg &
Blanc, 1981). Nevertheless, the decrease in the aw of the Cheddar
cheeses during ripening (w0.006 between 30 and 180 d) was lower
than that for either Emmental or Gruyère (w0.016 and 0.031,
respectively between 30 and 150 d), as estimated from the data of
Ruegg and Blanc (1981). The inter-variety differences in the magni-
tude of the decrease in aw during ripening probably reflect the
interactive effects of differences in contents of salt, moisture and
proteolysis products (Costa et al., 2010; Marcos et al., 1981; Ruegg &
Blanc, 1977). Moreover, the different methods used to determine aw
in the above studies may also contribute to the differences in the
magnitude of aw values reported for the different cheese varieties
(Leung, Morris, Sloan, & Labuza, 1976).
3.5. Relationship between aw and compositional parameters and
proteolysis
Linear regression analyses at individual ripening times indicated
that the correlation between aw and composition varied with both
Fig. 2. Change in water activity (aw) of experimental Cheddar cheese during ripening;
presented data show the values for 20 cheeses at 1 d and 32 cheeses at all other
ripening times.
compositional parameter and ripening time (Table 4). Most notable
was the significant correlation between aw and the level of salt-in-
moisture (S/M) at ripening times
30 d. This trend concurs with
that reported in the studies of López et al. (1990), which concluded
that the aw of young cheese was determined mainly by the salt-in-
moisture (S/M) content in the range 0.36e1.12%. In contrast, the aw
of the 270 d-old cheese correlated positively with contents
of moisture and moisture-in-non-fat substances (MNFS) but
negatively with fat content; the latter relationship is expected
owing to the significant inverse relationship (r ¼ 0.85; df ¼ 30)
between moisture and fat. No good explanation can be offered for
the changing relationship between aw and the levels of moisture, S/
M, MNFS and fat over ripening time. Tentative explanations may
include dynamic changes in the types and concentrations of dif-
ferent solutes (e.g., the decrease in L() lactate and increases in
FAAs and peptides) and their interactive effects on aw, especially
when the range of moisture content (35.6e38.6%, w/w) was rela-
tively small, for example compared to that found in brine-salted
cheese shortly after salting, e.g., from w19 to 46% in San Simón
cheese for which Marcos et al. (1983a) reported a significant pos-
itive relationship between aw and moisture content. Moreover,
changes in microbial dynamics and the concomitant changes in
substrate (solute) utilisation and production (Smit, Smit, & Engels,
2005) may also confound the effects of moisture on aw.
Linear regression analysis indicated that aw was positively
correlated with concentrations of lactose and L(þ)-lactate and
total lactate, and negatively with content of D() lactate when all
ripening times were included in the regression analysis (Table 5).
The inverse relationship between aw and D()-lactate may be
indicative of the indirect effect of proteolysis which, like D()-
lactate increases with ripening time as L(þ)-lactate is racemised
by growing NSLAB population (Lynch, McSweeney, Fox, Cogan, &
Drinan, 1996, 1997). An increase in D(þ) lactate per se, which
tends to precipitate in the form of insoluble calcium-lactate in-
clusions (Kubantseva, Hartel, & Swearingen, 2004), would be
expected to have little direct effect on aw; indeed, the concurrent
decreases in lactose and L(þ) lactate may be expected to increase
the aw. Hence, it would appear that the depressive effects on aw of
the increase in the level of peptides and FAAs in the moisture
phase with ripening time have a larger effect than the decrease in
the L(þ) lactate. A further contributory factor could be an increase
in water binding capacity of the casein associated with a chelation
of casein-bound calcium by the D()-lactate, similar to the hy-
dration of casein accompanying the removal of most of the
calcium from the para-casein by addition of emulsifying
salts (calcium chelating salts), such as sodium phosphates or cit-
rates during, the manufacture of processed cheese (Guinee &
O’Kennedy, 2012).
In the main, there were no relationships between these param-
eters and aw at individual ripening times, which may reflect the
narrow spread in the concentrations of these parameters at these
times. In contrast with the trend noted in the current study, analysis
 D.K. Hickey et al. / International Dairy Journal 30 (2013) 53e58 57

Table 4
Linear regression coefficients of relationship between water activity (aw) of experimental Cheddar cheeses and compositional parameters at different ripening times.a

Ripening time (d) df Moisture Fat Protein Salt S/M MNFS FDM
1 18 0.19ns 0.31ns 0.16ns 0.68** 0.74*** 0.05ns 0.33ns
30 30 0.30ns 0.33ns 0.21ns 0.26ns 0.36* 0.21ns 0.28ns
90 30 0.46** 0.36ns 0.06ns 0.07ns 0.20ns 0.43** 0.20ns
180 30 0.22ns 0.24ns 0.31ns 0.15ns 0.25ns 0.18ns 0.19ns
270 30 0.65*** 0.59*** 0.13ns 0.09ns 0.28ns 0.55** 0.39ns
a
Values are percentages; statistical significance of the linear regression coefficients is denoted by the superscripts: *p < 0.05; **p < 0.01; ***p < 0.001; ns (non significant),
p > 0.05. Abbreviations: df, degrees of freedom; S/M, salt in moisture; MNFS, moisture in non fat substance; FDM, fat in dry matter.

Table 5
Linear regression coefficients of relationship between water activity (aw) and pH, lactose, L(þ)- and D()-lactate, total lactate, pH 4.6 soluble N and total free amino acids at
different ripening times.a

Ripening time (d) df pH Lactose L(þ)-lactate D()-lactate Total lactate pH 4.6 soluble N Total FAAs
1 18 0.43ns 0.14ns 0.04ns 0.12ns 0.04ns 0.07ns 0.14ns
30 30 0.07ns 0.00ns 0.07ns 0.04ns 0.46* 0.03ns 0.30ns
90 30 0.32ns 0.09ns 0.11ns 0.42* 0.1ns 0.02ns 0.23ns
180 30 0.18ns 0.03ns 0.13ns 0.28ns 0.06ns 0.13ns 0.34ns
270 30 0.11ns 0.09ns 0.09ns 0.27ns 0.10ns 0.31ns 0.42*
Over ripening 146 0.22** 0.32*** 0.37*** 0.60*** 0.32*** 0.79*** 0.84***
a
Values are percentages except for total FAAs, which are in mg 100 g1; statistical significance of the linear regression coefficients is denoted by the superscripts: *p < 0.05;
**p < 0.01; ***p < 0.001; ns (non significant), p > 0.05. Abbreviations: df, degrees of freedom; TN; total N; FAAs, free amino acids.

of data of Marcos, Millan, Esteban, Alcala, and Fernández-Salguero when data for all ripening times were included but not when the
(1983b) from different Spanish cheese varieties indicated a strong data for individual ripening times was included only (data not
significant negative correlation (P < 0.001) between aw in retail shown). The former is expected as there was a significant
samples and lactic acid concentration which varied in the range of (P < 0.001) relationship between Pep X activity and FAA concen-
0.34e1.77% and moisture from 29.8 to 57.6% moisture. tration (r ¼ 0.36; df ¼ 98), which reduces aw (Fig. 2).
There was a significant, though weak, negative correlation be-
tween aw and pH when values for all ripening times were included in
the regression analysis (Table 5), but when values for specific rip-
ening times were included no correlation was observed. It is note-
worthy that Marcos et al. (1981) found no correlation between aw
and pH of 25 different cheese varieties that had pH values ranging
from 4.75 to 6.55. A stronger inverse relationship between aw and pH
may be expected when considering the effects of increasing pH on
the casein network, namely an increase in negative charge and,
hence, in its potential to bind water through groups, especially non-
protonated carboxyl groups of glutamic and aspartic acids. However,
such an effect may be confounded by other factors such as the
deposition of soluble calcium and phosphate in the form of insoluble
calcium phosphate complexes with the casein.
The concentrations of pH 4.6 soluble N and FAAs were most
strongly correlated with aw, with the latter decreasing as the con-
centrations of these water soluble compounds increased during
maturation (Fig. 3). The inverse relationship between aw and pro-
teolysis is expected as proteolysis involves hydrolysis of peptide
bonds and the expectant immobilization of water by the free amino
(NHþ 
3 ) and carboxyl (COO ) groups via hydrogen bonding.
Moreover, the release of FAAs and small peptides from the protein
may increase the potential of polar functional groups (e.g., COO,
NHþ 3 ) on FAAs and peptides to hydrogen bond with water
(Damodaran, 1997). The trends in aw with proteolysis are consistent
with those of Saurel et al. (2004) who reported that aw in Emmental
cheese over ripening was correlated with the concentration of free
NH2 groups. Previously, Marcos et al. (1981) found a significant
correlation (r ¼ 0.671, P < 0.001) between aw and the concen-
tration of non-protein nitrogen (soluble in 12% trichloro-acetic
acid; amino acids and peptides) in a range of retail cheese sam-
ples that included ‘soft’, semi-soft, semi-hard and hard varieties Fig. 3. Relationships between water activity (aw) and pH 4.6 soluble N (r ¼ 0.79,
df ¼ 146) and free amino acids (FAAs, r ¼ 0.84, df ¼ 146) in experimental Cheddar
varying widely in composition and pH. cheeses during ripening. Linear regression lines (e) were fitted to the experimental
Linear regression analysis of Pep X activities and aw indicated data in each case. The data were obtained from 20 cheeses at 1 d and 32 cheeses at all
a significant (P < 0.001) inverse relationship (r ¼ 0.33; df ¼ 98) other ripening times.
58 D.K. Hickey et al. / International Dairy Journal 30 (2013) 53e58
4. Conclusions
Changes in the aw of 32 experimental Cheddar cheeses with
compositions typical of those found in commercial Cheddar cheese
were measured over a 270 d ripening period. The aw of young
cheese (
30 d) was inversely correlated with S/M level but was
not influenced by variation in contents of fat, moisture-in-non-fat
substances, fat-in-dry-matter or pH. The aw of mature, 270-d old
cheese was positively correlated with levels of moisture, MNFS but
negatively with content of fat. The aw decreased significantly in all
cheeses during ripening, with the mean value for the 32 cheeses
decreasing from 0.964 at 1 d to 0.956 at 270 d. Linear regression
analysis of the dataset pertaining to all ripening times suggests
that the preponderant factor responsible for the decrease in aw was
the increases in proteolysis, as measured using pH 4.6 soluble N
and FAAs.
Acknowledgements
This work was funded by the Department of Agriculture, Fish-
eries and Food, under the National Development Plan and Food
Institutional Research Measure. We gratefully appreciate the sta-
tistical advice of Ms Ana-Maria Magdalina.
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