ARTICLE IN PRESS

International Dairy Journal 18 (2008) 129–138

www.elsevier.com/locate/idairyj

The effect of high-pressure treatment at 300 MPa on ripening

of ewes’ milk cheese
Bibiana Juan, Victoria Ferragut, Buenaventura Guamis, Antonio-José Trujillo
Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), CeRTA, XiT, Departament de Ciència Animal i dels Aliments,
Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
Received 26 April 2007; accepted 17 July 2007

Abstract

The effect of high-pressure (HP) treatment (300 MPa for 10 min) applied on day 1 or 15 of ripening on the proteolysis, lipolysis and
sensory characteristics of ewes’ milk cheese was investigated in real industry conditions (full-size cheese processed using an industrial HP
machine). HP treatment increased the hydrolysis of b-casein at all stages of ripening. Furthermore, at 90 days of ripening, higher
hydrolysis of as1-casein was found in HP-treated cheeses than in control cheeses. HP treatment applied on day 1 of ripening considerably
increased the production of free amino acids. However, this treatment significantly changed the final sensory characteristics of cheeses;
the texture of cheeses treated on day 1 became softer, more elastic and less crumbly, and the scores for taste, odour and aroma quality
decreased compared with control cheese. Cheeses HP-treated on day 15 of ripening were similar to control cheeses, but with a more
homogeneous protein network and a less crumbly texture; these cheeses had the highest percentage of short-chain fatty acids and were
preferred by the panel.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: High-pressure treatment; Ripening acceleration; Ewes’ milk cheese

1. Introduction McSweeney, Sendra, Kelly, and Guamis (2002), applying

The ripening of cheese involves a series of processes
necessary for the development of flavour and texture
characteristics. The biochemistry of cheese ripening in-
volves glycolysis, lipolysis and proteolysis, the last being
the principal and most complex biochemical event in most
cheese varieties (Fox, 1989). The ripening of cheeses is a
slow and consequently an expensive process that is not
fully predictable or controllable. Consequently, new
approaches have been attempted to accelerate it, including
the use of high-pressure (HP) (Law, 2001). Yokohama,
Sawamura, and Motobayashi (1992) filed a patent for
acceleration of cheese ripening using HP, which reported
that Cheddar cheese could be ripened in 3 days by
treatment at 50 MPa, in comparison to a 6 month ripening
period for conventional Cheddar cheese. O’Reilly, O’Con-
nor, Murphy, Kelly, and Beresford (2000) and Saldo,
Corresponding author. Tel.: +34 935813292; fax: +34 935812006.
E-mail address: toni.trujillo@uab.es (A.-J. Trujillo).
the same conditions in Cheddar and goat milk cheeses,
respectively, did not obtain proteolysis rates similar to
those suggested by the Japanese group. In Camembert
(Kolakowski, Reps, and Babuchowski, 1998; Reps,
Kolakowski, and Dajnowiec, 1998) and Père Joseph
cheeses (Messens, Foubert, Dewettinck, & Huyghebaert,
2000), an increase in proteolysis was observed by applying
50 MPa. Nevertheless, there was not a significant influence
on proteolysis of Kurpiowski cheeses (Reps et al., 1998)
and Gouda (Messens, Estepar-Garcı́a, Dewettinck &
Huyghebaert, 1999; Kolakowski et al., 1998; Reps et al.,
1998) at any HP treatment applied.
Other authors have studied the use of higher pressures
for shorter times. Saldo et al. (2002) HP-treated goat milk
cheeses at 400 MPa for 5 min and found twice the levels of
total free amino acids (TFAAs) in HP-treated cheeses
compared to the control. However, this HP treatment
decelerated lipolysis in cheese (Saldo et al., 2003). The
ripening acceleration of Mozzarella cheese was possible
with treatment at 200 MPa for 60 min, which produced

0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2007.07.005
 ARTICLE IN PRESS
130 B. Juan et al. / International Dairy Journal 18 (2008) 129–138
changes in the rheology, meltability and appearance,
producing cheeses similar to those matured conventionally
(Johnston & Darcy, 2000). The shredability of Cheddar
cheese was also accelerated by HP processing (345 or
483 MPa for 3 or 7 min) showing attributes similar to those
produced after 27 days of ripening (Serrano, Velázquez,
Lopetcharat, Ramı́rez, & Torres, 2004). In Hispánico
cheese manufactured with a mixture of cows’ and ewes’
milk, HP treatment at 400 MPa for 5 min accelerated the
hydrolysis of casein and increased the TFAA content, but
did not influence the taste quality and flavour intensity of
the cheese (Ávila, Garde, Gaya, Medina, & Núñez, 2006).
In conclusion, the use of HP in cheese ripening acceleration
requires the study of treatment conditions in each variety
of cheese.
The possibility of the acceleration of ripening in ewes’
milk cheese has previously been tested in our laboratories
using a range of pressures from 200 to 500 MPa for 10 min
on days 1 and 15 of ripening. Overall, treatment at
300 MPa produced the highest level of proteolysis in this
variety of cheese. Cheeses HP-treated at 300 MPa on day 1
of ripening presented the highest levels of TFAAs, whereas
those treated at the same pressure on day 15 of
manufacturing had the highest levels of water-soluble
nitrogen (WSN) (Juan, Ferragut, Guamis, Buffa, &
Trujillo, 2004; Juan, Ferragut, Buffa, Guamis & Trujillo,
2007b). In most of the studies cited, experiments were
performed using small pieces or portions of cheeses, which
were HP-treated using laboratory- or pilot-scale machines
(100 mL-5–L). Nevertheless, to obtain more conclusive
results, the treatment of whole commercial cheese
(real size) by means of the application in an industrial
machine is necessary, to test the potential of HP technology
in industrial application for acceleration of cheese ripening.
In the present study, proteolysis, lipolysis and sensory
characteristics of commercial cheeses (1.3 kg) treated at
300 MPa in an industrial-scale HP system were evaluated.
The effect of HP treatment on the volatile profile of
some of the cheeses in this study during aging has been
published separately (Juan, Barrón, Ferragut, Guamis, &
Trujillo, 2007a).
2. Material and methods
2.1. Cheesemaking and HP treatment
Three independent batches of commercial cheeses
(1.3 kg) were manufactured using a standard procedure
in a dairy farm (MontBru, Moià, Spain), as described
elsewhere (Juan et al., 2007b). Cheeses were HP-treated by
using a ACB horizontal HP unit with a 350 L capacity
(ACB, Nantes, France). One group of cheeses was treated
at 300 MPa for 10 min on the first day after cheese making
(3P1), whereas another group was treated at the same
conditions at 15 days of ripening (3P15). A third group of
cheeses, maintained in the ripening chamber, were used
as a control.
2.2. Cheese composition
Gross composition of cheeses was determined in grated
samples. Triplicate samples were assayed for total solids,
fat and total nitrogen as described elsewhere (Buffa,
Guamis, & Trujillo, 2005). The pH was measured with a
pH-meter (Micro-pH 2001, Crison, Alella, Spain) on a
cheese/distilled water (1:1) slurry.
Salt was determined by chloride analysis (Corning 926
Chloride Analyzer, Sherwood Scientific Ltd., Cambridge,
UK) after cutting cheeses in sectors to get three different
zones: external, medium and internal area.
2.3. Autolysis
Starter autolysis was assayed 24 h post-pressurization
by determination of lactate dehydrogenase (LDH) activity
as described by O’Reilly, O’Connor, Murphy, Kelly, and
Beresford (2002), and results expressed as international
units (U) g1 cheese, where one unit was defined as
the amount of enzyme that catalyses the reduction of
1 mmol of nicotinamide adenine dinucleotide (NAD) per
minute.
2.4. Measurement of WSN and free amino acids
Water-soluble extracts (WSE) of the cheeses, the pH 4.6-
soluble nitrogen (WSN) fraction, TFAAs and individual
free amino acids (FAAs) were prepared and analysed as
described by Buffa et al. (2005).
2.5. Capillary electrophoresis analyses
The water-insoluble fractions recovered during the
extraction of WSE were washed three times with 1 M
sodium acetate buffer (pH 4.6) and the remaining fat was
eliminated by washing with dichloromethane–sodium
acetate buffer (1:1, v/v). The final protein precipitate was
then lyophilized. Capillary electrophoresis (CE) analyses
were performed following the method of Recio and
Olieman (1996). Separations were carried out using an
Agilent CE Instrument (Agilent Technologies, Waldbronn,
Germany) controlled by Chemstation Software (Agilent).
The separations were performed using a fused-silica
capillary column (BGB Analytik, Essen, Germany) of
0.6 m  50 mm i.d. (effective length 0.53 m) applying voltage
of 20 kV at 45 1C and a final current of around 33 mA.
Sample injection was performed at a pressure of 50 mbar
for 4 s. Protein components were detected at 214 nm and
the area of each peak was integrated using Agilent
ChemStation Operation Software.
The breakdown of caseins in the HP-treated and con-
trol cheeses was examined by determination of the
amount of intact casein at each sampling time during
ripening and expressed as a percentage of the respective
area at day 1.
 ARTICLE IN PRESS
B. Juan et al. / International Dairy Journal 18 (2008) 129–138
2.6. Peptide analysis by HPLC
Peptides in the water-soluble fraction were separated by
reversed-phase high performance liquid chromatography
(HPLC) using an automated system (LCM1; Waters
Corporation, Milford, MA, USA). Separations were
carried out on a 250  4.6 mm column packed with
C18-bonded silica gel with a particle diameter of 5 mm
and pore width of 30 nm (Simmetry 300TM, Waters) at a
constant temperature of 40 1C, following the method of
González del Llano, Polo, & Ramos (1995). After running
the samples, the integration area of peptides, excluding that
of FAAs, was determined. The area of peptides eluted
between 10 and 35 min was considered the hydrophilic
peptide area, whereas the area of peptides eluting between
35 and 80 min was considered the hydrophobic peptide
area. The amounts of hydrophobic and hydrophilic
peptides were expressed as units of chromatogram area
per mg of cheese dry matter.
2.7. Free fatty acid analysis
Free fatty acids (FFAs) were extracted according to the
modified method of De Jong and Badings (1990). Cheese
(1 g) was placed in a screw-capped tube, and ground with
3 g anhydrous Na2SO4, 0.3 mL H2SO4 (2.5 M) and 30 mL
internal standard solution (heptanoic acid 37.3 mg mL1).
Diethyl ether–heptane (3 mL; 1:1, v/v) was added and the
mixture was shaken for 3 min using a vortex mixer. The
supernatant was transferred to a screw-capped tube
containing 1 g anhydrous Na2SO4. This operation was
repeated three times. The lipid extract was applied to an
aminopropyl column Spe-ed NH2 500 mg mL1 (Applied
Separations, Allentown, PA, USA), which was conditioned
with 10 mL heptane. Hexane/2-propanol (20 mL; 3:2, v/v)
was applied to eliminate glycerides, and FFAs were eluted
with 5 mL diethyl ether containing 2% formic acid. A
direct injection of this solution (1 mL) was used for gas
chromatographic analysis. For each sample, two extrac-
tions of FFAs were carried out, and two chromatographic
injections were made for each extract.
Gas chromatography was carried out with a Hewlett
Packard (HP) (Agilent Technologies) chromatograph
(HP 6890), equipped with an automatic on-column
injector. A flame-ionization detector (FID) was used either
with a fused silica capillary column, 30 m  0.25 mm i.d.,
coated with TRB-FFAP phase (df ¼ 0.25 mm). The carrier
gas was high-purity helium at flow rates of 0.9 mL min1.
High-purity hydrogen (40 mL min1) and compressed air
(450 mL min1) were supplied to the FID. Temperature
was raised from 50 to 240 1C at 5 1C min1 and then
held at 240 1C for 20 min. The output signal from the
detector was integrated using HP 6890 ChemStation
software. Individual FFAs were identified and quantified
using standards supplied by Sigma (St. Loius, MO, USA).
Standard solutions with increasing concentrations of
individual fatty acids and fixed concentrations of internal
131
standards were used for the calculation of calibration
curves.
2.8. Texture and colour determination
Uniaxial compression test was applied using a TA-TX2
Texture Analyzer (State Micro system, Survey, UK), as
described by Juan, Trujillo, Guamis, Buffa, and Ferragut
(2007c).
The colour was evaluated using a portable Hunterlab
spectrocolorimeter (MiniscanTM XE, Hunter Associates
Laboratory, Reston, VA, USA) with a cool white
fluorescent (Fcw) illuminant and observer at 101. The
hunterlab scale (L, a, b) was used where ‘‘L’’ is a measure
of the lightness and ranges between 0 and 100. Parameter
‘‘a’’ varies from green () to red (+), and ‘‘b’’ varies from
blue () to yellow (+). Total colour differences of 3P1 and
3P15-cheeses referred to control cheese (DE) were calcu-
lated using formula:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
DE ¼ DL2 þ Da2 þ Db2 . (1)
Measurements were taken on different points of the
surface of cheeses cut in two halves. Eight consecutive
measures were taken for every cheese.
2.9. Sensory analysis
Sensory assessments of 30 and 90 day-old cheese samples
were carried out by 20 panellists from the Centre Especial
de Recerca Planta de Tecnologia dels Aliments (CERPTA)
who were experienced in the sensory assessment of cheeses.
Panellists were asked to give a score on a 1–7 point scale
(1, low; 4, medium; 7, high) for the following taste (quality,
saltiness, acidic, bitterness, sharpness and after taste) and
texture (hardness, elasticity and crumbliness) attributes.
The overall acceptability of cheese was evaluated on a 1–7
point scale (1, defective; 3, acceptable; 5, good; 7,
excellent). A preference test also was made, where the
tasters had to choose between two samples of cheese
(control and 3P1, control and 3P15, 3P1 and 3P15).
Sensory assessment was performed at room temperature.
Water and bread biscuits were delivered to neutralize
the taste between the different cheese samples during
assessment.
2.10. Statistical analysis
Data were processed by analysis of variance (ANOVA)
using the general linear models (GLM) procedure of SASR
System (SAS Institute, Inc., Cary, NC 27513, USA). The
Student-Newman–Keuls test was used for comparison of
sample data. Level of significance was set at Po0.05. The
results of the sensory preference test were analysed
according to the tables of Roessler, Baker, and Amerine
(1956).
 ARTICLE IN PRESS
132 B. Juan et al. / International Dairy Journal 18 (2008) 129–138

3. Results and discussion 3.2. Effect of HP treatment on proteolysis in cheese

3.1. Effect of HP treatment on cheese composition Residual levels of as1-casein decreased considerably
during ripening in all cheeses during ripening, whereas
Cheeses had a mean level of 35.09% and 57.9% protein b-casein was hydrolysed to a lesser extent (Fig. 1). This low
and fat, respectively, both expressed on % of dry matter degree of breakdown of b-casein is typical for ewes’ milk
basis. HP-treated cheeses had better water retention cheese, in which b-casein is very resistant to proteolysis
properties compared with control cheeses (Table 1), (Marcos, Fernández-Salguero, & Esteban, 1978). At 90
showing higher moisture content than untreated cheeses days of ripening, control cheeses had higher residual as1
at the end of ripening, agreeing with the results previously and b-casein levels than HP-treated cheeses, respect to the
reported (Juan et al., 2007b). Similar results were obtained amounts on the first day. At 15 days of ripening, 3P1-
in Père Joseph (Messens et al., 2000) goat’s milk cheese due cheeses had higher levels of residual as1-caseins than
to HP-induced changes in the cheese protein network,
forming a new structure that better retains the water in Table 2
Salt-in-moisture content (means7standard error)a of control and high-
cheeses. Cheeses HP-treated on day 1 of ripening had the pressure (HP)-treated ewes’ milk cheeses during ripening
highest pH values (Table 1) at 15, 30 and 60 days of
ripening and this could be related to the decrease in the Salt-in-moisture at ripening time (days)
starter counts observed after HP treatment (Juan et al.,
1 15 60
2007b), responsible for lactose fermentation and lactic acid
production. Cheeses HP-treated on the 15 days of ripening Exterior
had similar pH values to control cheeses, indicating that Control 2.7370.39 2.7470.09 3.8570.21a
the acidification process was completed at the initial stages 3P1b 2.5870.35 2.7470.08 3.4670.38b
3P15 2.7470.05 4.0170.25a
of ripening. No differences in pH values were found
between cheeses at the end of ripening. Medium
The salt-in-moisture content was higher in HP-treated Control 1.1870.44 2.5170.07b 3.9370.16c
3P1 1.2870.33 2.7170.04a 4.1870.34b
than control cheeses at 15 and 60 days of ripening 3P15 2.6570.06a 4.3370.30a
(Table 2). A faster salt diffusion was observed in HP-
treated than in control cheeses, as HP-treated cheeses Interior
Control 0.4870.22 2.1470.09b 3.8970.14b
showed higher levels of salt-in-moisture content in the 3P1 0.4870.26 2.3270.10a 4.3170.15a
medium and interior sectors than control cheeses at 15 and 3P15 2.2370.21ab 4.4270.15a
60 days of ripening, respectively. These results agreed with
Total
those reported by Saldo et al. (2002) for goats’ milk cheeses Control 2.0070.09 2.5870.18b 3.9770.32b
HP-treated at 50 MPa for 72 h and 400 MPa for 5 min. A 3P1 1.9670.07 2.7870.27a 4.0870.58a
more continuous microstructure has been described in HP- 3P15 2.8370.11a 4.2370.09a
treated cheeses (Capellas, Mor-Mur, Trujillo, Sendra, & a
Means in the same column and area of cheese followed by a different
Guamis, 1997; O’Reilly et al., 2003; Torres-Mora et al., superscript letter differ (Po0.05).
1996), which could facilitate the diffusion of salt from the b
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15,
rind to the core. cheeses HP-treated at 300 MPa at 15 days of ripening.

Table 1
Moisture content and pH values (means7standard error)a of control and high-pressure (HP)-treated ewes’ milk cheese during ripening

Ripening time (days)

1 15 30 60 90

Mb(%)
Control 48.2470.25a 42.8870.42 39.4771.43 34.1471.1 28.9971.60b
3P1c 47.4770.44b 42.2972.94 39.0572.53 35.6670.26 33.2373.06a
3P15 40.0371.65 38.9571.15 33.5170.16 30.7371.37a
PH
Control 5.4170.41 4.9270.04c 4.8670.04b 4.8270.09b 4.8570.12
3P1 5.3670.05 5.2570.01a 5.1570.16a 4.9870.29a 4.9870.29
3P15 4.9570.04b 4.9470.08b 4.8870.10ab 4.8670.12
a
Means within the same column and parameter followed by different superscript letter differ (Po0.05).
b
M, Moisture content.
c
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15, cheeses HP-treated at 300 MPa at 15 days of ripening.

B. Juan et al. / International Dairy Journal 18 (2008) 129–138
100
80
Residual α s1-CN (%)
60
40
20
0 5
0 20 40 60
90
Residual β -CN (%)
70
50
0 20 40 60
Ripening time (days)
Fig. 1. Residual levels of as1- and b-casein, expressed as a percentage of
the amount in the corresponding 1-day-old cheeses, in control (K), 3P1
(&) and 3P15 (n) ewes’ milk cheese throughout the ripening.
control and 3P15-cheeses. However, hydrolysis of
as1-casein was significantly higher in HP-treated cheeses
than control cheeses at 90 days of ripening. O’Reilly et al.
(2000) reported a significant decrease in the levels of intact
as1-casein in Cheddar cheese HP-treated at 50 MPa
for 72 h, and attributed this to conformational changes
produced in the casein matrix by the pressure, which may
render the proteins susceptible to the action of proteases.
Hydrolysis of b-casein was significantly higher in HP-
treated cheeses than control cheeses. At 90 days of
ripening, 3P1-cheeses had lower levels of residual b-casein
than 3P15-cheeses, and were also lower than those of the
control. An increase of the hydrolysis of b-casein was also
observed in Cheddar cheese treated at 200–400 MPa at
25 1C for 20 min (O’Reilly et al., 2002) and in ewes’ milk
cheese treated at 200–500 MPa at 12 1C for 10 min (Juan
et al., 2007a), which was attributed to conformational
changes in the paracasein gel structure, which lead to the
exposure of susceptible peptide bonds from b-casein that
are readily cleaved by plasmin.
The highest amount of WSN at 15 days of ripening was
found in 3P15-cheeses (Fig. 2). However, no significant
differences were found between cheeses at 60 and 90 days
of ripening. The breakdown of peptides produced by
primary hydrolysis of caseins is caused by the enzymes
from lactic acid bacteria and non-starter microorganisms,
producing short water-soluble peptides and amino acids.
ARTICLE IN PRESS
133
25
20
WSN/TN (%)
15
10
80 100 0 30 60 90
12
TFAA (mg Leu g-1)
4
0
0 30 60 90
Ripening time (days)
80 100
Fig. 2. Levels of water-soluble nitrogen (WSN), and total free amino-
acids (TFAA) in control (K), 3P1 (&) and 3P15 (n) ewes’ milk cheese
throughout ripening.
In general, the lowest levels of hydrophobic peptides and
the lowest ratio of hydrophobic to hydrophilic peptides
were found in 3P1-cheeses (Table 3). These results could be
related to the release of intracellular peptidases that
hydrolyse the hydrophobic peptides into low molecular
weight peptides and FAAs, in agreement with the highest
TFAAs levels found in 3P1-cheeses (Fig. 2). Pressure can
increase the autolysis of starter culture cells, accelerating
the peptidolytic reactions (Crow, Martley, Coolbear, &
Roundhill, 1995), which influence the rate of secondary
proteolysis in cheese. The highest level of autolysis
occurred in 3P1-cheeses which showed the highest LDH
activity (0.39 U g1 respect 0.27 and 0.30 of those of
control and 3P15-cheeses, respectively). According to
Malone, Shellhammer, and Courtney (2002) cell suspen-
sions of Lactococcus lactis subsp. cremoris MG1363 treated
at 300 MPa lysed significantly more than those treated at
100, 200, and 600–800 MPa for 5 min.
Cheese treated at 300 MPa at day 1 showed the highest
levels of TFAAs throughout ripening, and the highest
levels of the main individual FAAs (Fig. 3). HP-treated
cheeses only had lower levels of Arg and gamma-
aminobutyric acid (GABA) than control cheeses. There
are no reports suggesting that GABA has a direct or
indirect impact on cheese flavour. However, Arg is linked
to a bitter taste (Kirimura, Shimizu, Kimizuka, Ninomiya,
& Katsuya, 1969).
At 15 days of ripening, the TFAA levels found in 3P1-
cheeses were more than twice than those of control cheeses.
Also, the TFAA levels in 3P1-cheeses at 30 days of ripening
 ARTICLE IN PRESS
134 B. Juan et al. / International Dairy Journal 18 (2008) 129–138

Table 3
Hydrophobic and hydrophilic peptides in the water-soluble fractions of control and high-pressure (HP)-treated ewes’ milk cheese throughout the ripeninga

Ripening time (days)

1 15 30 60 90

Hydrophobic
Control 14177302 30407451a 35087517b 41307498a 38687620a
3P1b 14487300 20137354a 26507365b 32687670a 30517778b
3P15 30627593a 30317194b 40877112a 42597309a
Hydrophilic
Control 64731 228789 314796a 5447135 6157182
3P1 70739 235776 3017104a 4697193 5777151
3P15 216777 2507112b 4877170 6207150
Ratio
Control 26.7713 16.278.8a 12.675.6a 8.272.9 6.972.6
3P1 27.3714.5 9.874.7b 9.974.2b 7.873.2 5.371.1
3P15 15.976.7a 14.275.8a 9.272.9 7.271.8
a
Values are means of duplicate determinations in three cheese-making experiments, expressed as % area mg1 dry matter. Means within the same
column followed by different superscript letter are significantly different (Po0.05).
b
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15, cheeses HP-treated at 300 MPa at 15 days of ripening.

1400

1200
FAA (mg kg-1 dry matter)

1000

800

600

400

200

0
IT

IS
L
P
R
R
N
LU
LN

LY

A
O

YS

U
ET

S
E

G
AM A

O I
N
VA

IL
AL
AS

LY
PH

AB
LE
TH
SE
AS

TY

R
PR

AR
C

H
O
G
G
G

M
C

Fig. 3. Levels of individual free fatty acids, (FAAs) (expressed as mg FAA kg1 of dry matter) in control (&), 3P1 ( ) and 3P15 ( ) ewes’ milk cheese at
90 days of ripening.

were higher than those of control cheeses at 60 days of Table 4

ripening. At 60 days, 3P1-cheeses presented higher levels of Free fatty acid (FFA) profile (means7standard error)a of control and
TFAAs than those of control cheese at 90 days. Using the high-pressure (HP)-treated ewes’ milk cheese at 60 and 90 days of ripening
amino acid content as an index of cheese ripening, the
Days FFA Totalc
possibility of reducing cheese ripening time by more than 1
month in 60- or 90-day old cheeses by HP treatment at SCFAb (%) MCFA (%) LCFA (%)
300 MPa applied on the first day could be possible.
Control 60 20.0570.70b 19.3371.92ab 60.872.39ab 1.2870.27a
90 20.3472.08b 19.0571.08 6073.11a 1.5170.15a
3.3. Effect of HP treatment on lipolysis in cheese 3P1d 60 17.9270.62c 20.4470.64a 61.670.36a 0.9970.19b
90 18.5972.59b 19.370.84 60.672.68a 1.1970.07b
Lipolysis releases FFAs that contribute directly to cheese 3P15 60 22.077 2.12a 18.870.73b 59.172.23b 1.3270.22a
flavour, especially short- and intermediate-chain FFAs 90 22.771.83a 19.470.54 57.871.3b 1.5270.19a
(Collins, McSweeney, & Wilkinson, 2003). Cheese treated a
Means within the same column and day followed by different
at 300 MPa at day 1 had lower amounts of FFAs than superscript letter are significantly differentP(Po0.05). P
control and 3P15-cheeses at 60 and 90 days of ripening b
Short-chain fatty acids (SCFA, %) ¼ (C4:0C8:0)/ (C4:0C18:2) 
P P
(Table 4). In a previous unpublished study, working with 100, medium-chain fatty acids (MCFA, %) ¼ (C10:0C14:0)/ (C4:0
P
ewes’ milk cheese HP-treated at 200–500 MPa on days 1 C
P18:2)  100, long-chain fatty acidsP (LCFA, %) ¼ (C16:0C18:2)/
(C4:0 C18:2)  100, total FFA ¼ (C4:0C18:2).
and 15 of ripening, 3P1-cheeses showed the highest degree c
Total (mg  g1 fat).
of lipolysis at 1 and 15 days of ripening, which was d
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15,
attributed to the faster and better interaction of microbial cheeses HP-treated at 300 MPa at 15 days of ripening.
 ARTICLE IN PRESS
B. Juan et al. / International Dairy Journal 18 (2008) 129–138 135

lipases with the substrate due to the lysis produced by HP
treatment. A possible explanation for the lower levels of
FFAs obtained for 3P1-cheeses in the present study could
be attributed to a higher metabolism of FFAs to other
aromatic compounds compared to control and 3P15-
cheeses. FFAs can react with alcohols to yield esters
(McSweeney & Sousa, 2000). In agreement with these
results, higher amounts of ethyl esters were found in ewes’
milk cheeses HP-treated at 300 MPa on the first day of
ripening, compared to those treated at 15 days of ripening
and untreated cheeses (Juan et al., 2007b).
At 90 days of ripening, the highest percentages of short-
chain FFAs were found in 3P15-cheeses, which also
showed the highest levels of butyric, caproic, caprilic,
lauric and myristic acids (results not shown).
3.4. Effect of HP treatment on sensory characteristics of
cheese
Fracture stress can be used as a fracturability index (low
numerical value indicates greater fracturability), and
fracture strain describes the deformability of cheese (higher
numerical value indicates greater deformability). The
modulus values may express the stiffness of cheeses (Zoon,
1991) and higher values indicate that cheese exhibits a
higher resistance to deformation.
Fracture stress and the modulus of deformability
increased, and the fracture strain decreased, during cheese
ripening (Table 5). Cheese treated at 300 MPa at day 1
showed the highest fracture strain and the lowest modulus
values in all ripening stages (Table 5), becoming the most
deformable cheeses. Moreover, the lowest facture stress
values were found in 3P1-cheeses at 90 days of ripening.
Moisture content, pH and proteolysis of casein affect the
texture of cheeses. As a result of proteolysis, the casein
matrix becomes less strong and decreases the force
necessary for the rupture of cheeses (Bertola, Bevilacqua,
& Zaritzky, 1991, 1992). In addition, the increase in
moisture content would also contribute to a cheese that
deformed more easily. At 90 days of ripening, 3P1-cheeses
had higher moisture content and hydrolysis of as1-casein
than control and 3P15-cheeses, and this could explain the
softening of 3P1-cheeses. The last one also had higher pH
values than control and 3P15-cheeses at 15, 30 and 60 days
of ripening (Table 1). Visser (1991) reported that cheese
fracture stress decreases on increasing the pH from 4.8 to
5.2. At pH 5.2 the protein–protein bonds in cheese are at
their weakest; consequently, the casein particles in the
cheese matrix are more deformable (Visser, 1991).
In terms of colour, HP-treated cheeses had significantly
lower lightness values than control cheeses (Table 6) and
3P1-cheeses had lower lightness than 3P15-cheeses. In
general, HP-treated cheeses showed higher yellowness values
than control cheeses. The decrease in lightness and increase
in yellowness found in HP-treated cheeses have also been
observed in goat milk fresh cheeses treated at 500 MPa for 5,
15 or 30 min at 10 1C (Capellas, Mor-Mur, Sendra, &
Guamis, 2001) and Garrotxa cheeses treated at 50 MPa for
72 h (Saldo, Sendra, & Guamis 2001); this was related to
structural changes induced by HP treatment. A more
continuous protein network with regular distribution of
hole-size has been described in fresh (Capellas et al., 1997)
and hard goat cheeses (Saldo et al., 2001) as a consequence
of HP treatment. Also, the protein phase in Cheddar cheese
was more continuous after pressure treatment at 350 MPa
for 70 h (O’Reilly et al., 2003) and 483 MPa for 7 min
(Serrano, Velazquez, Lopetcharat, Ramirez, & Torres, 2005)
than in untreated cheeses. The changes produced in the
protein matrix by pressure could explain the colour
differences obtained in HP-treated cheeses. Total colour
difference (DE) values relative to the control cheese were
higher for 3P1-cheeses than those of 3P15.

Table 5
Uniaxial compression parameters in control and high-pressure (HP)-treated ewes’ milk cheese throughout the ripeninga

Ripening time (days)

1 15 30 60 90

Fracture stress (  10 kPa)

Control 5.7970.66b 8.8570.92a 10.6771.20 14.6972.38 20.8173.8a
b
3P1 7.1671.97a 9.7375.12a 9.4572.38 1474.30 15.9473.51b
3P15 7.0870.97b 10.7671.20 13.7272.91 21.674.5a
Fracture strain ()
Control 0.7070.06b 0.2870.02c 0.2470.02b 0.2170.03b 0.1870.02b
3P1 0.7670.05b 0.6370.11a 0.4670.06a 0.3270.06a 0.2770.05a
3P15 0.3470.10b 0.2670.03b 0.2270.06b 0.1870.01b

Modulus (  10 KPa)
Control 10.8373.45a 47.95714.75a 66.93726.72a 92.84755.5 125.51768.84ab
3P1 7.9573.46b 16.03711.16c 27.3714.37b 76.47744.05 94.72741.83b
3P15 29.61711.82b 59.39731.42a 80.65758.01 150.25770.85a
a
Values are means7standard error. Means within the same column followed by different superscript letter are significantly different (Po0.05).
b
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15, cheeses HP-treated at 300 MPa at 15-days of ripening.
 ARTICLE IN PRESS
136 B. Juan et al. / International Dairy Journal 18 (2008) 129–138

Table 6
Colour measurementsa of control and high-pressure (HP)-treated ewes’ milk cheese throughout the ripening

Ripening time (days)

1 15 30 60 90

L
Control 93.4271.29a 92.1670.75a 91.9971.03a 90.0470.91a 88.3870.97a
3P1b 92.6071.55b 89.7971.39c 88.8871.02c 86.4071.57c 85.6172.04c
3P15 90.4971.16b 90.7771.16b 88.0271.39b 86.6971.25b
a (–)
Control 0.6670.10 0.5070.24 0.4370.22 0.3170.28 0.1270.31a
3P1 0.7370.20 0.5870.25 0.4670.29 0.3770.31 0.2470.35b
3P15 0.5570.23 0.3970.23 0.3570.23 0.0570.27a

b
Control 8.9971.02b 9.5871.49b 9.8871.42 10.5471.58b 11.7171.94
3P1 10.0370.97a 11.4271.87a 11.5773.15 13.1271.40a 12.7772.13
3P15 10.9372.02a 10.5571.81 12.2171.72a 12.2571.83
DE
3P1 1.49 3.77 3.88 3.87 1.85
3P15 2.88 1.61 1.60 1.68
a
Means within the same column followed by different superscript letter are significantly different (Po0.05).
b
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15, cheeses HP-treated at 300 MPa at 15 days of ripening.

Table 7
Sensory characteristics (scale 1–7)a of control and high-pressure (HP)-treated ewes’ milk cheese at 30 and 90 day of ripening

Flavour Texture Global impression

Acid Salty Bitter Sharp After-taste Taste Aroma Odour Elasticity Crumbliness Hardness
quality quality quality

Day 30
Control 4.7a 4.04a 3.14 3.41 2.9 4.33 4.31a 4.43a 3.31b 4.16a 3.95 4.42a
3P1b 3.26b 3.22b 2.74 3 3.15 4.08 3.73b 3.61b 4.73a 3.14b 3.85 3.38b
3P15 4.2a 4.05a 3.02 3.59 2.59 4.54 4.41a 4.47a 3.55b 3.65ab 3.66 4.58a
Day 90
Control 4.11 4.02 3.13 3.75 2.99 4.89a 4.44a 4.70a 2.50c 5.06a 4.15 4.78a
3P1 3.58 3.73 3.1 3.69 3.44 4.13b 3.84b 3.55b 4.68a 3.12c 3.91 3.64b
3P15 3.93 4.09 3.19 3.66 3.08 5a 4.73a 4.87a 3.02b 4.46b 4.12 4.76a
a
Means within the same column and day followed by different superscript letter are significantly different (Po0.05).
b
3P1, cheeses HP-treated at 300 MPa on the first day of ripening; 3P15, cheeses HP-treated at 300 MPa at 15 days of ripening.

Cheese treated at 300 MPa at day 1 received the lowest
taste, aroma and odour quality scores from the panellists
(Table 7). The lower odour and aroma quality could be
attributed to the lower percentage of short-chain FFAs
found in 3P1-cheeses. Fernández-Garcia, López-Fandiño,
Alonso, & Ramos (1994) asserted that the liberation of
short-chain fatty acids seemed to be desirable for flavour
fullness and balance in Manchego-type cheese. In addition,
the flavour effect of FFAs in cheese is regulated by pH; in
cheeses with a high pH, the flavour effect of FFAs may be
negated due to neutralization (Molimard & Spinnler,
1996). Cheese treated at 300 MPa at day 1 showed the
highest pH values at 15, 30 and 60 days of ripening.
At 30 days of ripening, 3P1-cheeses showed the lowest
scores for acid taste in agreement with the higher pH found
in these cheeses. Scores for salty taste were also lower in
3P1-cheeses than control and 3P15-cheeses at 1 month old,
which could be explained by the more homogeneous
distribution of salt observed in these cheeses. At the end
of ripening, both acid and salty tastes were similar in all
cheeses. Nevertheless, in spite of 3P1-cheeses having the
lowest content of hydrophobic peptides and the lowest
ratio of hydrophobic:hydrophilic peptides, the panel did
not evaluate these cheeses as significantly lower in
bitterness. Additionally, the highest levels of TFAAs found
in 3P1-cheeses were not associated with a stronger flavour
development. The concentration of TFAAs is not con-
sidered to be directly responsible for cheese flavour
(Broome, Krause, & Hickey, 1990), the rate-limiting factor
being the conversion of amino acids to aroma compounds
 ARTICLE IN PRESS
B. Juan et al. / International Dairy Journal 18 (2008) 129–138
(Fox & Wallace, 1997). It is possible that HP treatment
applied on the first day of ripening affected the enzymes
involved in amino acid biochemistry and subsequent
compound formation in cheese. In the previous study of
volatile compounds, 3P1-cheeses had lower amounts of
aldehydes, ketones, short-chain fatty acids and terpenes
than control and 3P15-cheeses (Juan et al., 2007b).
Cheese treated at 300 MPa at day 1 were defined as the
most elastic and least crumbly cheeses, which could be
explained by changes in the casein network and the water
content as previously described; overall, 3P1-cheeses were
scored lowest by the panel. When panellist had to choose
between two different cheeses, most of the panellists choose
3P15-cheeses respect to control cheeses, nevertheless,
differences were not statistically significant. However,
statistically significant (Po0.05) differences were found
when panellists had to choose between cheeses HP-treated
at two different stages of ripening, 3P15-cheeses being
more preferred than 3P1-cheeses by the panellists.
4. Conclusions
Treatment of ewes’ milk cheese at 300 MPa for 10 min
created a cheese matrix with better water retention
properties with a faster diffusion of salt from the rind to
core. This HP treatment had a marked effect on cheese
proteolysis; hydrolysis of as1-CN and b-CN was enhanced
by the pressure. The levels of secondary proteolysis
depended on when pressure was applied. The highest
TFAA levels as well as the highest rate of cell lysis were
observed in 3P1-cheeses. From TFAA results, a reduction
of more than 1 month ripening time was observed in
3P1-cheeses, as the same degree of ripening was obtained
after 26 and 58 days of ripening instead of 60 and 90 days,
respectively. However, this treatment significantly changed
the sensory characteristics of cheeses; a new texture
developed which was more elastic, softer and less crumbly.
This HP treatment had important consequences in taste
and aroma of cheeses, showing that amino acid catabolism
plays a major role in aroma formation in cheese. Cheeses
HP-treated at day 15 of ripening had the highest values of
short-chain FFAs, but displayed values between those of
control and 3P1-cheeses for most of the other variables
studied. 3P15-cheeses were given lower crumbliness and
higher elasticity scores than control cheeses, but higher and
lower, respectively, than 3P1-cheeses. Comments of the
panellists indicated that these cheeses had the greatest
flavour and the best texture; hence, they were the favourites
of the panellists.
Acknowledgements
The authors acknowledge the Comisión Interministerial
de Ciencia y Tecnologı́a for the financial support given to
this investigation (CICYT AGL2000-1426-C02). Juan, B.
acknowledges a predoctoral fellowship from the Comissio-
nat per a Universitat i Recerca de la Generalitat de
137
Catalunya. We would acknowledge the use of the Espuña
S.A. high pressure facilities in Olot (Spain).
References
Ávila, M., Garde, S., Gaya, P., Medina, M., & Núñez, M. (2006). Effect of
high-pressure treatment and a bacteriocin-producing lactic culture on
the proteolysis, textura, and taste of Hispanico cheese. Journal of Dairy
Science, 89, 2882–2893.
Bertola, N. C., Bevilacqua, A. E., & Zaritzky, N. E. (1991). Changes in
rheological and viscolelastic proporties and protein breakdown during
the ripening of ‘‘Port Salut Argentino’’ cheese. International Journal of
Food Science and Technology, 26, 467–478.
Bertola, N. C., Bevilacqua, A. E., & Zaritzky, N. E. (1992). Proteolytic
and rheological evaluation of maturation of Tybo Argentino cheese.
Journal of Dairy Science, 75, 3273–3281.
Broome, M. C., Krause, D. A., & Hickey, M. W. (1990). The use of non-
starter lactobacilli in Cheddar cheese manufacture. Australian Journal
of Dairy Technology, 45, 67–73.
Buffa, M., Guamis, B., & Trujillo, A. J. (2005). Specific effect of high-
pressure treatment of milk on cheese proteolysis. Journal of Dairy
Research, 72, 385–392.
Capellas, M., Mor-Mur, M., Sendra, E., & Guamis, B. (2001). Effect of
high-pressure processing on physico-chemical characteristics of fresh
goats’ milk cheese (Mató). International Dairy Journal, 11, 165–173.
Capellas, M., Mor-Mur, M., Trujillo, A. J., Sendra, E., & Guamis, B.
(1997). Microstructure of high pressure treated cheese studied by
confocal scanning light microscropy. In K. Heremans (Ed.), High
pressure research in the biosciences and biotechnology (pp. 391–394).
Leuven, Belgium: Leuven University Press.
Collins, Y., McSweeney, P. L. H., & Wilkinson, M. G. (2003). Lipolysis
and free fatty acid catabolism in cheese: A review of current
knowledge. International Dairy Journal, 13, 841–866.
Crow, V. L., Martley, F. G., Coolbear, T., & Roundhill, S. J. (1995). The
influence of phage-assisted lysis of Lactococcus lactis subsp. lactis ML8
on Cheddar cheese ripening. International Dairy Journal, 5, 451–472.
De Jong, C., & Badings, H. T. (1990). Determination of free fatty acids in
milk and cheese. Procedures for extraction, clean up and capillary gas
chromatographic analysis. Journal of High Resolution Chromatogra-
phy,, 13, 94–98.
Fernández-Garcia, E., López-Fandiño, R., Alonso, L., & Ramos, M.
(1994). The use of lipolytic and proteolytic enzymes for the
manufacture of Manchego-type cheese from bovine and ovine milk.
Journal of Dairy Science, 77, 2139–2149.
Fox, P. F. (1989). Acceleration of cheese ripening. Food Biotechnology, 2,
133–185.
Fox, P. F., & Wallace, J. M. (1997). Formation of flavour compounds.
Advances in Applied Microbiology, 45, 17–85.
González del Llano, D., Polo, M. C., & Ramos, M. (1995). Study of
proteolysis in artisanal cheeses: high performance liquid chromato-
graphy of peptides. Journal of Dairy Science, 78, 1018–1024.
Johnston, D. E., & Darcy, P. C. (2000). The effects of high pressure
treatment on immature Mozzarella cheese. Milchwissenschaft, 55,
617–620.
Juan, B., Barron, L. J. R., Ferragut, V., Guamis, B., & Trujillo, A. J.
(2007b). Changes in the volatile composition of a semi-hard ewe milk
cheese induced by high-pressure treatment of 300 MPa. Journal of
Agricultural and Food Chemistry, 55, 747–754.
Juan, B., Ferragut, V., Buffa, M., Guamis, B., & Trujillo, A. J. (2007a).
Effects of high pressure on proteolytic enzymes in cheese: Relationship
with the proteolysis of ewe milk cheeses. Journal of Dairy Science, 90,
2113–2125.
Juan, B., Ferragut, V., Guamis, B., Buffa, M., & Trujillo, A. J. (2004).
Proteolysis of high pressure-treated ewe’s milk cheese. Milchwis-
senschaft, 59, 616–619.
Juan, B., Trujillo, A. J., Guamis, V., Buffa, M., & Ferragut, V. (2007c).
Rheological, textural and sensory characteristics of high-pressure
 ARTICLE IN PRESS
138 B. Juan et al. / International Dairy Journal 18 (2008) 129–138
treated semi-hard ewes’ milk cheese. International Dairy Journal, 17,
248–254.
Kirimura, J., Shimizu, A., Kimizuka, A., Ninomiya, T., & Katsuya, N.
(1969). The contribution of peptides and amino acids to the
taste of food-stuffs. Journal Agricultural and Food Chemistry, 17,
689–695.
Kolakowski, P., Reps, A., & Babuchowski, A. (1998). Characteristics of
pressurised ripened cheeses. Polish Journal of Food and Nutrition
Sciences, 7, 473–482.
Law, B. A. (2001). Controlled and accelerated cheese ripening: The
research base for new technologies. International Dairy Journal, 11,
383–398.
Malone, A. S., Shellhammer, T. H., & Courtney, P. D. (2002). Effects of
high pressure on the viability, morphology, lysis, and cell wall
hydrolase activity of Lactococcus lactis subsp. cremoris. Applied and
Environmental Microbiology, 68, 4357–4363.
Marcos, A., Fernández-Salguero, J., & Esteban, M. A. (1978). Hidrólisis
relativa de las caseı́nas del queso tipo Manchego maduro y primeros
productos de su degradación proteolı́tica. Archivos de Zootecnia, 27,
341–350.
McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways for
the production of flavour compounds in cheese during ripening. A
review. Lait, 80, 293–324.
Messens, W., Estepar-Garcia, J., Dewettinck, K., & Huyghebaert, A.
(1999). Proteolysis of high-pressure-treated Gouda cheese. Interna-
tional Dairy Journal, 9, 775–782.
Messens, W., Foubert, I., Dewettinck, K., & Huyghebaert, A. (2000).
Proteolysis of a high-pressure-treated smear-ripened cheese. Milchwis-
senschaft, 55, 328–332.
Molimard, P., & Spinnler, H. E. (1996). Review: Compounds involved in
the flavour of surface mold-ripened cheeses: Origins and properties.
Journal of Dairy Science, 79, 169–184.
O’Reilly, C. E., O’Connor, P. M., Murphy, P. M., Kelly, A. L., &
Beresford, T. P. (2000). The effect of exposure to pressure of 50 MPa
on Cheddar cheese ripening. Innovative Food Science and Emerging
Technologies, 1, 109–117.
O’Reilly, C., O’Connor, P. M., Murphy, P. M., Kelly, A. L., & Beresford,
T. P. (2002). Effects of high-pressure treatment on viability and
autolysis of starter bacteria and proteolysis in Cheddar cheese.
International Dairy Journal, 12, 915–922.
O’Reilly, C., Kelly, A. L., Oliveira, J. C., Murphy, P. M., Auty, M. A. E.,
& Beresford, T. P. (2003). Effect of varying high-pressure treatment
conditions on acceleration of ripening of Cheddar cheese. Innovative
Food Science and Emerging Technologies, 4, 277–284.
Recio, I., & Olieman, C. (1996). Determination of denatured serum
proteins in the casein fraction of heat-treated milk by capillary zone
electrophoresis. Electrophoresis, 17, 1228–12133.
Reps, A., Kolakowski, P., & Dajnowiec, F. (1998). The effect of high
pressure on microorganims and enzymes of ripening cheeses. In N. S.
Isaacs (Ed.), High pressure food science, bioscience and chemistry
(pp. 265–270). Cambridge, UK: Royal Society of Chemistry.
Roessler, E. B., Baker, G. A., & Amerine, M. A. (1956). One-tailed and
two-tailed tests in organoleptic comparisons. Food Research, 21,
117–120.
Saldo, J., Fernández, A., Sendra, E., Butz, P., Tauscher, B., & Guamis, B.
(2003). High pressure treatment decelerates the lipolisis in a caprine
cheese. Food Research International, 36, 1061–1068.
Saldo, J., McSweeney, P. L. H., Sendra, E., Kelly, A. L., & Guamis, B.
(2002). Proteolysis in caprine milk cheese treated by high pressure to
accelerate cheese ripening. International Dairy Journal, 12, 35–44.
Saldo, J., Sendra, E., & Guamis, B. (2001). Hard cheese structure after a
high hydrostatic pressure treatment at 50 MPa for 72 h applied to
cheese alter brining. Lait, 81, 625–635.
Serrano, J., Velazquez, G., Lopetcharat, K., Ramı́rez, J. A., & Torres, J.
A. (2004). Effect of moderate pressure treatments on microstructure,
texture and sensory properties of stirred-curd of Cheddar shreds.
Journal of Dairy Science, 87, 3172–3182.
Serrano, J., Velazquez, G., Lopetcharat, K., Ramirez, J. A., & Torres, J.
A. (2005). Moderately high hydrostatic pressure processing to reduce
production costs of shredded cheese: Microstructure, texture and
sensory properties of shredded milled curd Cheddar. Journal of Food
Science, 70, 286–293.
Torres Mora, M. A., Soeldner, A., Ting, E. Y., Hawes, A. C. O., Aleman,
G. D., Bakshi, G. S., et al. (1996). Early microstructure changes in
Cheddar cheese and the effects of high pressure curd processing. In
IFT annual meeting. New Orleans, Lousiana, USA Book of abstracts
(p. 9).
Visser, J. (1991). Factors affecting the rheological and fracture properties
of hard and semi-hard cheese. In: Bulletin of the International Dairy
Federation (pp. 49–61). Brussels, Belgium: International Dairy
Federation.
Yokohama, H., Sawamura, N., & Motobayashi, N. (1992). Method for
accelerating cheese ripening. European Patent application EP, 469,
857(0) A1.
Zoon, P. (1991). The relation between instrumental and sensory
evaluation of the rheological and fracture properties of cheese. In
Bulletin of the International Dairy Federation, (pp. 30-35). Brussels,
Belgium: International Dairy Federation.