doi: 10.1111/1471-0307.

12156

ORIGINAL
RESEARCH

Effects of Penicillium roqueforti and whey cheese on

gross composition, microbiology and proteolysis of
mould-ripened Civil cheese during ripening
SONGUL CAKMAKCI,1 ALI A HAYALOGLU,2* ELIF DAGDEMIR,1
B U L E N T C E T I N , 1 M U S T A F A G U R S E S 1 and D E R E N T A H M A S KAHYAOGLU1
1

Department of Food Engineering, Ataturk University, 25240 Erzurum, Turkey, and 2Department of Food
Engineering, Inonu University, 44280 Malatya, Turkey

Four different types of mould-ripened Civil cheese were manufactured. A dened (nontoxigenic)
strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without
addition of the whey cheese (Lor); in parallel, secondary starter-free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The
incorporation of whey cheese in the manufacture of mould-ripened Civil cheese altered the gross
composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds
appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments
during the rst 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were
observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in
the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 12 log cfu/g towards the end of ripening.
Keywords Blue-type cheese, Mould-ripened Civil cheese, Penicillium roqueforti, Proteolysis.

INTRODUCTION

*Author for
correspondence. E-mail:
adnan.hayaloglu@inonu.
edu.tr
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594

Civil cheese has been produced for years in eastern

Turkey, comprising Erzurum and neighbouring
cities, based on the spontaneous growth of mould
present in the ripening environment. Civil cheese
has a Protected Geographical Indication status, and
its characteristics are specied in the documents
published by Turkish Patent Institute (TPI 2009).
Due to its low fat level, its popularity has increased
recently among people with high cholesterol or
similar health problems (Cambaztepe et al. 2009).
To produce Civil cheese, skimmed cow milk is
allowed to rest overnight at about 1520 C to
increase the acidity by the action of the natural
microora. Sometimes, the acidity of skim milk
may be increased with the addition of acidied
whey (until 0.5% of lactic acid). Skim milk is
heated to 30 C, and calf rennet is added for
coagulation. As the heating process is slowly
increased with a slow agitation, the curd forms at

about 5052 C. Curd particles are encouraged to

adhere to the ladle during the process of mixing
until the temperature reaches about 5560 C.
The cheese block is transferred onto a clean stainless steel table by a paddle and kneaded by hand.
The cheese block is then hung from a platform to
stretch by its own weight. The stretching process
is repeated until the curd has a smooth, plastic
and brous character. The manufacturing protocol
and gross chemical composition have been extensively described by Cambaztepe et al. (2009),
TPI (2009) and Cakmakci (2011). After this
cheese is produced, it may be ripened in different
forms. The cheese is ripened in brine (1012%
NaCl) or matured as dry salted together with/
without Lor cheese either in goat skins or in a
hardened plastic barrel. In dry-salted cheeses,
moulds grow spontaneously on or within the
brous structure of the cheese during ripening
and the unique avour of the cheese is developed
late in the ripening period. So, the resultant

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cheese is evaluated as a different variety and named mouldy

Civil cheese. In this situation, mouldy Civil cheese is completely different from regular Civil cheese; many microbiological and biochemical changes occur during ripening. This
mouldy Civil cheese is very popular in the eastern region of
Turkey. The ripened cheese has a blue greenish colour, and a
stringy structure (Cakmakci et al. 2012a, 2013).
The quality of cheese is inuenced by the type of milk,
manufacturing technology and complex changes during
ripening, caused by indigenous milk enzymes, the coagulant
used, starter cultures, nonstarter bacteria and secondary
microora (Fox et al. 1996; McSweeney 2004). The secondary microora includes yeasts (e.g. Geotrichum candidum,
Debaryomyces hansenii), moulds (e.g. Penicillium roqueforti,
Penicillium camemberti) and bacteria (e.g. Corynebacterium
spp., Micrococcus spp., Propionibacterium spp. and heterofermentative lactobacilli), and they have signicant effects on
the ripening of cheeses (Chamba and Irlinger 2004). The ripening of blue-type cheese varieties involving the growth of
surface moulds is more complex than with other varieties of
cheeses due to the broad enzyme systems of fungi. Growth of
Penicillium spp. in this type of cheese results in faster casein
breakdown, the formation of free amino acids and fat hydrolysis during ripening (Hayaloglu et al. 2008, 2011). Uncontrolled mould growth may cause potential hazards for
consumers (Cakmakci et al. 2012a). Due to increased interest
in this cheese, the fungal ora of mould-ripened Civil cheese
was determined by Cakmakci et al. (2012b), and the majority
(88.7%) of the fungal ora constituted various strains of
P. roqueforti. Despite the predominance of the genus in
mould-ripened Civil samples, nothing has been published in
the literature on the effect of the mould on cheese proteolysis.
To the best of our knowledge, there is also no information
related to proteolysis of mouldy Civil cheese, except our survey study. In our previous survey study, we determined that
soluble nitrogen fractions (WSN/TN, TCA-SN/TN and PTASN/TN) values changed widely (respectively, 5.227.2%,
4.9014.93%; 2.455.31%) (Cakmakci et al. 2012a).
Use of a dened strain of P. roqueforti and incorporation
with whey cheese (called Lor in Turkey) to ll the openings
within the brous structure in the manufacture of the cheese
and observing the chemistry, microbiology and biochemistry
of cheese during ripening would contribute to a better
understanding of the cheese. The aim of this study was the
production of mould-ripened Civil cheese with and without
Lor addition and use of P. roqueforti spores and to investigate the chemistry, microbiology and ripening of the
cheeses during 180 days.
MATERIALS AND METHODS

Cheesemaking procedure
Experimental mould-ripened Civil cheese was manufactured
as described on the ow sheet (Figure 1). Skimmed cows
2014 Society of Dairy Technology

milk (500 L) and Lor cheese were supplied from a local

dairy plant (Leben Dairy Factory, Co., Inc., Erzurum, Turkey). To produce Lor cheese, the extra whey was boiled,
and the resulting coagulated matter was broken up into tiny
pieces. Whey used for acidication of skim milk was
obtained from remains of the previous days Civil cheese
production. Penicillium roqueforti (SC 509, nontoxigenic),
which was previously isolated from mouldy Civil cheese,
was identied by partial sequencing ITS1-5.8S-ITS (Cakmakci et al. 2012b) and was inoculated at a level of
104 cfu/mL. Commercial calf rennet (180 IMCU/mL) was
obtained from Mayasan Company Inc., Istanbul, Turkey.
Hardened plastic containers (supplied from a local rm,
Erzurum, Turkey), which are suitable for food packaging,
were used for packaging of cheeses. Holes with 4 mm
diameter were available to allow air entry as shown in
Figure 2. The proximate composition of acidied skim milk,
Lor cheese and whey used in Civil cheese production is
shown in Table 1. The same procedure in cheesemaking
was followed as described in Cakmakci et al. (2013). The
resultant cheeses were ripened at 4  1 C sampled after 2,
30, 60, 90 or 180 days.

Gross chemical and microbiological analyses

Cheese samples were analysed in duplicate for moisture
using the oven-drying method at 102 C (IDF 1982), fat by
the Van Gulik method (Ardo and Polychroniadou 1999)
and total nitrogen by the micro-Kjeldahl method (IDF
1993). For pH measurement, 10 g of sample was macerated
in 10 mL of distilled water and the pH of the resultant
slurry was measured using a digital pH meter (pH 211,
Microprocessor pH Meter; Hanna Inst., Rome, Italy). Titratable acidity (% lactic acid) was measured as outlined by
AOAC (1995). For microbiological analyses, 10 g of
cheese was weighed and dispersed aseptically in 90 mL of
saline peptone water (ISO 6887) including 0.85% NaCl
plus 0.1% peptone and homogenised (eight strokes per second) in a sterile polyethylene bag using a Stomacher (Mayo
HG400 Stomacher, Baranzate, Milano, Italy) for 2 min. Serial
dilutions were made in saline peptone water (ISO 6887)
(Harrigan 1998). Total aerobic mesophilic bacteria were
enumerated on plate count agar (Merck, Darmstadt, Germany)
at 30  1 C for 48 h (Harrigan 1998), psychrotrophic bacteria on plate count agar (Merck) at 7  1 C for 10 days
(Harrigan 1998), enterococci on kanamycin aesculin azide
agar (Merck) at 36 C  1 for 2448 h (Harrigan 1998),
lactobacilli on MRS agar (Merck) at 32  1 C for 35 days
under anaerobic conditions (Harrigan 1998), lactococci on
M17 agar (Merck) at 30  1 C for 24 or 48 h (Gilliland
et al. 1984) and yeasts and moulds on potato dextrose agar
(Merck) at 25 C for 5 days (Harrigan 1998). The mould
counts were separately determined in the same media (PDA
medium) considering the macroscopic and microscopic
appearance.
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Raw cows milk

Cream separation
Gradual heating to 45-50 C
Addition of acidified whey by indigenous microorganisms (ca. 0.5%, as lactic acid)
Addition of rennet (180 IMCU/mL) with gentle stirring
Gradual heating to 5560 C with stirring
Transfer the curd onto a table and kneading
Hang the curd on a platform for stretching and to form a fibrous texture (20 C)
Rest for overnight and pressing for about 2 days (20 C)
The following day, shred the block like string
Shredded curd was divided into two parts

Add Lor cheese (25%, w/w)

and uniform completely

Add coarse salt (3%)

Add coarse salt (3%)

Filling into the 1 kg of containers
and ripening for 6 mo
Inoculation of P. roqueforti spores
104 cfu/mL

Inoculation of P. roqueforti spores

104 cfu/mL

Filling into the 1 kg of containers

and ripening for 6 mo

Filling into 1 kg of containers

and ripening for 6 mo

Filling into 1 kg of containers

and ripening for 6 mo

KL cheese

4S cheese

4L cheese

KS cheese

Figure 1 A ow sheet for the production of experimental mould-ripened Civil cheese. Control (KS), control with whey cheese (KL); 4S, inoculated
with Penicillium roqueforti at a level of 1 9 104 cfu/mL and without Lor cheese; 4L, inoculated with P. roqueforti at a level of 1 9 104 cfu/mL and
with 25% addition of Lor cheese.

Figure 2 Mould-ripened Civil cheese after addition of Penicillium roqueforti spores. A few holes are opened by a needle on the lid of container to
ensure air entry. Arrows show holes.

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where the effect of treatments (four samples), time (ve

ripening periods) and replicates (three trials) were estimated for response variables. Duncans multiple-comparison test was used as a guide for pair comparisons of
treatment means using the SPSS package programme version 9.0 for Windows (SPSS Inc., Chicago, IL, USA). The
level of signicance of differences between treatments was
determined at P < 0.05.

Table 1 The proximate composition of acidied skim milk, Lor

cheese and acidied whey
Materials

Total solid (%)

Fat (%)

Protein (%)

pH

Acidied skim milk

Lor cheese
Acidied whey

8.97
30.35
5.41

0.12
4.00
0.10

3.79
21.86
0.98

5.23
5.10
3.13

Proteolysis
Water-soluble nitrogen (WSN), 12% trichloroacetic acidsoluble nitrogen (TCA-SN) and 5% phosphotungstic acidsoluble nitrogen (PTA-SN) fractions were determined by the
methods described in Hayaloglu et al. (2011) and expressed
as a percentage of total nitrogen in the cheese. The waterinsoluble fractions of the cheeses were freeze-dried and then
analysed by urea-polyacrylamide gel electrophoresis (ureaPAGE) using a Protean II XI vertical slab gel unit (Bio-Rad
Laboratories Ltd., Watford, UK) according to the method of
Andrews (1983), and the gels were stained directly using
the method of Blakesley and Boezi (1977) with Coomassie
Brilliant Blue G-250. The WSN fractions of the cheeses
were also freeze-dried and analysed by RP-HPLC as
described by Hayaloglu et al. (2011).
Statistical analysis
Data from chemical composition and microbiological
counts were analysed by analysis of variance (ANOVA) for
each ripening time point (2, 30, 60, 90 or 180 days). ANOVA
was performed with a general linear model procedure,

RESULTS AND DISCUSSION

Microbiology
The microbiological results are presented in Table 2. Averaged values of total bacteria (8.30 log cfu/g), lactobacilli
(7.80 log cfu/g), lactococci (8.27 log cfu/g), psycrotrophs
(8.13 log cfu/g) and yeasts and moulds (7.60 log cfu/g)
were determined at the beginning of the ripening. The same
values were observed by Gobbetti et al. (1997) and Seratlic
et al. (2011) for Gorgonzola cheeses.
Counts for total bacteria, lactic acid bacteria (lactobacilli
and lactococci) and psycrotrophs decreased during ripening,
and the counts for these groups of bacteria were signicantly inuenced by the ripening time (P < 0.01). The
results agree with those reported by Florez et al. (2006) for
Cabrales cheese and Seratlic et al. (2011) for Gorgonzolatype cheese. Counts for total bacteria differed between the
samples, and their counts decreased steadily during the ripening. The numbers of total aerobic mesophilic bacteria
were lower in the cheeses containing added whey cheese
Lor (KL or 4L), reecting the point that heat treatment in
the production of Lor may reduce the microora of the

Table 2 Effect of treatments and storage periods on microbiology of mould-ripened Civil cheeses (log cfu/g)

Samples
KS
KL
4S
4L
Ripening time (day)
2
30
60
90
180
Source
Samples (S)
Ripening time (RT)
S 9 RT

Total aerobic
mesophils

Lactobacilli

Lactococci

Enterococci

Psychrotrophs

Yeasts and
moulds

Moulds

8.32
7.80
8.17
7.63






0.49a
0.59b
0.41a
0.70b

6.71
6.78
6.71
6.62






1.09a
0.73a
0.62a
0.76a

7.75
7.60
7.56
7.75






0.91a
0.67a
0.89a
0.99a

4.60
4.47
4.45
4.48






0.28a
0.15a
0.23a
0.18a

8.32
7.95
8.16
7.87






0.52a
0.49bc
0.37ab
0.45c

7.89
7.03
7.96
8.03






0.53a
0.58b
0.42a
0.41a

2.09
2.18
5.71
5.80






1.79b
1.83b
1.56a
1.18a

8.30
8.30
8.04
7.93
7.33







0.30a
0.49a
0.56ab
0.61b
0.54c

7.80
7.15
6.53
6.26
5.78







0.38a
0.41b
0.27c
0.25d
0.51e

8.27
8.49
7.77
7.06
6.47







0.43a
0.24a
0.26b
0.60c
0.39d

4.42
4.49
4.48
4.56
4.53







0.24a
0.17a
0.17a
0.26a
0.24a

8.13
8.22
8.24
7.96
7.82







0.46a
0.47a
0.43a
0.52ab
0.47b

7.60
7.93
7.85
7.60
7.66







0.33a
0.36a
0.82a
0.58a
0.86a

2.00
2.46
2.83
5.66
6.80







1.12e
1.66d
2.03c
1.19b
0.86a

ANOVA

**
**
**

NS
**
**

NS
**
NS

NS
NS
NS

**
*
**

**
NS
**

**
**
**

KS, Civil cheese (control); KL, Civil cheese plus Lor cheese blends (75:25); 4S, Civil cheese inoculated with Penicillium roqueforti spores; 4L,
Civil cheese plus Lor cheese blends (75:25) inoculated with P. roqueforti spores; NS; nonsignicant. Means in the same row having different
letters are signicantly different (P < 0.05). *P < 0.05, **P < 0.01.

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cheese. The addition of Lor or P. roqueforti during production did not change the counts of lactobacilli, lactococci and
enterococci. Lactic acid bacteria exhibited a similar trend,
and their population decreased during ripening (P < 0.01);
however, the enterococci population did not change during
ripening to any signicant degree. Psycrotroph counts differed among the treatments, and their numbers had
decreased after 90 days of ripening. The yeast and mould
populations were similar in all the cheeses, with the exception of KL, and there were no signicant changes in the
population during ripening. The mould counts of 4L and 4S
samples were signicantly higher than the others. Higher
counts of moulds in 4L and 4S cheeses may be explained
by the inoculation of P. roqueforti in these cheeses. The
counts of mould (lamentous fungi) increased in all samples
during the storage period of 180 days. From the above
results it is apparent that, inoculation of P. roqueforti did
not change the count of bacteria in general (Table 2).

Chemical composition and pH

Mean values (SD) for total solids, protein, fat, acidity, salt
content (%) and pH of mould-ripened Civil cheeses are presented in Table 3. Effects of treatment and ripening time
were signicantly inuenced by the gross chemical composition and pH of the cheese. Total solid contents ranged
from 42% to 47% in the samples, and the values increased
during the ripening (P < 0.01). The highest values were
noted for the cheeses at 180 days of ripening (Table 3). The
lower level of fat in cheeses KS and 4S was noticeable, and
this was because they were produced from skimmed milk
and while Lor whey cheese was added to treatments KL
and 4L. Protein contents were higher than those reported for

other blue-type cheeses (Madkor et al. 1987; Zarmpoutis

et al. 1996; Fernandez-Salguero 2004), due to the low fat
level (<3%). The values ranged between 32% and 37%, and
its concentration increased signicantly after 90 days of ripening. The addition of Lor cheese increased the fat levels in
mould-ripened Civil cheeses; however, fat levels did not
change during ripening. The pH values of the cheeses
increased during ripening, and the sharp increases were
recorded at 180 days of ripening for all samples. The
increase in pH can be linked to the catabolism of organic
acids including lactic acid by moulds and/or production of
alkaline compounds such as ammonia and amines by protein
breakdown as discussed by other authors (Gobbetti et al.
1997; Hayaloglu et al. 2008; Seratlic et al. 2011).
Reported pH values for some other blue cheeses are
from 4.5 to 5.0 for Stilton (Madkor et al. 1987) and from
5.2 to 5.3 for Gorgonzola (Gobbetti et al. 1997), Picon
Bejes-Tresviso (Prieto et al. 1999) and Gorgonzola-type
cheese (Seratlic et al. 2011) in the rst week of ripening.
However, the pH of these cheeses increased towards the
end of the ripening period. In addition, it was reported
that commercial blue cheeses have pH values above 6.0
(Madkor et al. 1987; Zarmpoutis et al. 1996; FernandezSalguero 2004; Hayaloglu et al. 2008). The chemical
composition of the cheese was within the range reported
by Cambaztepe et al. (2009) for Civil cheeses; however,
the pH values found here were lower than those reported
by Cambaztepe et al. (2009). Although the levels of total
solids and fat were lower than those found in previous
survey of mould-ripened Civil cheese (Cakmakci et al.
2012a), the titratable acidity, protein and salt contents and
pH were similar.

Table 3 Effect of treatments and storage periods on some chemical composition and pH of mould-ripened Civil cheese samples
Samples

Total solid (%)

Protein (%)

Fat (%)

KS
KL
4S
4L
Ripening (day)
2
30
60
90
180
Source
Samples (S)
Ripening time (RT)
S 9 RT

44.99
42.10
45.23
42.94






2.63c
1.29a
1.77c
2.89b

34.65
31.93
36.47
32.56






2.18b
1.23a
1.62c
2.59a

1.00
2.60
1.00
2.56






0.00a
0.27b
0.00a
0.23b

41.60
42.79
42.94
44.76
46.97







1.64a
1.65b
1.42b
1.95c
2.21d

32.24
32.85
32.83
35.04
36.55







2.19a
2.14a
2.05a
2.14b
2.16c

1.69
1.85
1.89
1.79
1.72







0.73a
0.90a
0.95a
0.83a
0.77a

Aciditya (%)

pH

0.50
0.60
0.56
0.48






0.14a
0.14c
0.11b
0.18a

5.50
4.56
5.11
5.13






0.77c
0.15a
1.21b
1.17b

7.68
6.02
5.72
6.31






0.48d
0.44b
0.66a
0.59c

0.45
0.52
0.42
0.56
0.72







0.14a
0.13b
0.09a
0.09b
0.08c

4.60
4.59
4.79
4.92
6.48







0.15a
0.33a
0.40b
0.75b
1.17c

6.09
6.13
6.22
6.51
7.21







0.71a
0.77a
1.07a
0.94b
0.71c

Salt (%)

ANOVA

**
**
*

**
**
*

**
NS
*

**
**
**

**
**
**

**
**
**

KS, Civil cheese (control); KL, Civil cheese plus Lor cheese blends (75:25); 4S, Civil cheese inoculated with Penicillium roqueforti spores; 4L,
Civil cheese plus Lor cheese blends (75:25) inoculated with P. roqueforti spores; NS, nonsignicant. Means in the same row having different letters are signicantly different (P < 0.05). aExpressed as % of lactic acid. *P < 0.05, **P < 0.01.

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(a) 50

WSN (as % of TN)

40
30
20
10
0
2

30

60

90

30

60

90

30

60

90

180

TCA-SN (as % of TN)

(b) 25
20
15
10
5
0
180

(c) 10
PTA-SN (as % of TN)

Proteolysis
Proteolysis in mould-ripened Civil cheeses was estimated by
determining WSN, TCA-SN or PTA-SN fractions, RPHPLC analysis of peptides and urea-PAGE analysis of caseins during ripening. Total nitrogen values ranged from
5.00% to 5.71%, and differences in the total nitrogen levels
in the cheeses were signicant (P < 0.01) (data not shown).
The levels of total nitrogen increased signicantly after
90 days of ripening, reecting an increase in the total solids
(Table 2). The use of Lor cheese in the production proportionally decreased the levels of total nitrogen in cheese due
to lower total solids content in Lor (~30%) in comparison
with Civil cheese (~45%). The levels of soluble nitrogen
fractions (WSN, TCA-SN or PTA-SN) in the cheeses were
unchanged during the rst 60 days of ripening, with the
exception of KS. A sharp increase (Figure 3) in soluble
nitrogen values in KS cheese after 90 days of ripening was
unexpected due to absence of added fungal spores.
To our knowledge, mould growth can occur in Civil
cheese after a period (90 days), due to the impingement of
strings in the some cavities within the brous structure
(Cakmakci et al. 2013). The levels of proteolysis in KS
cheese were found to be higher than in the other cheeses.
Accordingly, the counts of mould and yeasts (in total
counts, see Table 2) were at the same levels as in 4S and
4L cheeses, which were inoculated with P. roqueforti
spores. The addition of Lor cheese lled the cavities within
the cheeses (see Figure 2), and the proteolysis indices and
the mould and yeast counts were found to be lower in comparison with the KS cheese. The use of Lor cheese in the
manufacture of mould-ripened Civil cheese resulted in a
similar effect on other proteolysis measurements, including
TCA-SN and PTA-SN. However, the level of WSN in the
cheese lled by Lor and inoculated with mould spores (4L)
increased. The levels of WSN increased signicantly over
the 180-day ripening (P < 0.01), and inoculation with
P. roqueforti signicantly (P < 0.01) inuenced the levels
of WSN. Penicillium-inoculated cheeses (samples of 4S and
4L) have 10-fold higher WSN values than KL cheeses at
180 days of ripening. More than 40% of the total nitrogen
in cheeses KS, 4S and 4 L was soluble in water at the end
of the ripening, but <10% of the nitrogen in the KL cheese
was water soluble. A high level of proteolysis was found
when the moulds became visible on the cheese, after
1 month of ripening, as described in Zarmpoutis et al.
(1996). The mould, P. roqueforti, is rich in proteolytic
enzymes (extracellular proteases) and contributes to the formation of certain peptides and amino acids (Cantor et al.
2004). The levels of TCA-SN and PTA-SN followed a similar trend during ripening of mould-ripened Civil cheeses
(Figure 3). The 12% TCA-SN fractions indicates the levels
of small peptides (between two and 20 amino acid residues),
free amino acids, ammonia and other minor compounds,

8
6
4
2
0
180

Ripening period (days)

Figure 3 Changes in water-soluble nitrogen (a), TCA-SN (b) and PTASN (c) fractions during the ripening. KS, KL, M 4S, 4L. See the
Materials and Methods section for abbreviations.

while 5% PTA-SN indicates peptides and amino acids of

molecular weights <600 Da (Jarrett et al. 1982; Hayaloglu
et al. 2011). These fractions indicate the level of secondary
proteolysis in cheese. According to the statistical analysis,
there was a signicant effect for treatments (use of
P. roqueforti spores and Lor cheese in production) and ripening time. No signicant differences were determined up
to 60 days of ripening, however as the ripening proceeded,
rapid increases both in TCA-SN and PTA-SN in the cheeses
were observed. The highest values for the two responses
were found in the KS cheese at 180 days of ripening. The
reason for this unexpected result may be growth of adventitious fungi, which was visible both on the surface and
inside of the brous structure of the KS cheese towards the
end of ripening.
Urea-PAGE electrophoretograms of the cheeses are presented in Figure 4. Hydrolysis of caseins appeared to be

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Figure 4 Urea-polyacrylamide gel electrophoresis electrophoretograms of the water-insoluble fractions of the cheeses during the ripening. See the
Materials and Methods section for abbreviations.

considerably slower in mould-ripened Civil cheeses during

the rst 90 days of ripening compared with other blue-type
cheeses (Cantor et al. 2004). Slight degradations (both
as1- and b-caseins) were observed after 90 days of ripening
in the KS cheese; however, both caseins were still intact in
other cheese samples (Figure 4). Both as1- and b-caseins
were extensively degraded after 180 days of ripening in the
all cheeses, with the exception of the KL cheeses. Compared with the other cheeses, little degradation took place
in KL cheese during the ripening, and also no changes were
noted in the levels of pH or soluble nitrogen fractions of
the KL cheese during ripening. Almost complete degradation of caseins in KS cheeses had taken place after
180 days of ripening, and only low concentrations of peptides were visible on the gel (Figure 4). Differences
between the control KS cheese and the other treatments
were also observed for pH (Table 3) and nitrogen fractions
(Figure 3). It was notable that the pH of KS cheese was
higher than those of other experimental cheeses (data not
shown), and a pH (>6.5) is favourable for plasmin action
(Hayaloglu et al. 2008). The 4S and 4L cheeses inoculated
with P. roqueforti underwent extensive degradation as highlighted on the gel pattern. Bands with lower electrophoretic
mobility begin to appear after 180 days of ripening, and
these originated from b-casein degradation, probably
through the action of plasmin and enzymes from P. roque600

forti. The mould strain has a metalloproteinase, aspartate

proteinase and alkaline proteinase (Gripon 1993), and the
metalloproteinase has an optimum activity for casein hydrolysis at pH 5.5, which is a common pH for most blue
cheeses (Cantor et al. 2004). Therefore, extensive and complex proteolysis due to different sources of proteolytic
enzymes occurred in mould-ripened Civil cheese as in other
blue cheeses, including Stilton (Madkor et al. 1987), Gorgonzola (Gobbetti et al. 1997), Picon Bejes-Tresviso (Prieto
et al. 1999), Cabrales (Florez et al. 2006) and Gorgonzolatype cheese (Seratlic et al. 2011).
RP-HPLC of the WSN fractions of the cheeses after 30,
60, 90 and 180 days of ripening is shown in Figure 5.
A number of peaks were eluted at early retention times
(especially 1328 min) for all ripening time points. These are
hydrophilic peptides, and the same kinds of peptides were
also observed for Turkish white-brined cheese by Hayaloglu
et al. (2004, 2005), for Tulum by Hayaloglu et al. (2007)
and for Malatya cheeses by Hayaloglu et al. (2010). Peptide
proles of the cheeses remained unchanged during the rst
90 days of ripening (with the exception of KS at 90 days);
however, sharp increases were seen in the peptides peaks
eluted at retention times of 4560 min. after 180 days of ripening (Figure 5). These are mainly hydrophobic peptides,
which can be fractionated using 70% ethanol. Hayaloglu
et al. (2005) showed that hydrophobic peptides insoluble in
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Day 30

KS

KL
4S
4L

Day 60

KS

KL
4S

Absorbance, at 214 nm

4L

Day 90

KS

KL
4S
4L

Day 180

KS

KL

4S
4L

10

20

30

40
Retention time, min

50

60

70

Figure 5 RP-HPLC peptide proles of water-soluble fractions of the cheeses during the ripening. See the Materials and Methods section for abbreviations.

2014 Society of Dairy Technology

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70% ethanol were eluted at retention times of 4560 min

under identical chromatographic conditions. The use of heat
(6062 C) in the manufacture of Civil cheese may inactivate residual coagulant and heat-labile enzymes of microbial
or milk origin. The effect of P. roqueforti spores was relatively low until 90 days of ripening. Slow growth of
P. roqueforti on the KL and 4L cheeses may be linked to
the blending with Lor cheese (it has a granular texture) at a
level of 25%. A brous structure in Civil cheese allows the
growth of fungi during ripening; however, Lor cheese lled
the cavities within the brous structure. Qualitative and
quantitative differences were observed among cheese manufactured using Lor (KL and 4L) and without Lor (KS and
4S) at all ripening time points (Figure 5). The water-soluble
prole followed the same trend in the cheese samples during
the ripening treatment with added; Lor cheese had a lower
level of proteolysis than Lor cheese-free samples (Figure 3).
CONCLUSIONS
The levels of proteolysis found in mould-ripened Civil
cheeses were about half the level that has been reported
for commercially available blue-type cheeses. Distinctive
features of the mould-ripened Civil cheese were lower
levels of fat and higher levels of salt compared with
other blue-type cheeses. As the high level of salt slowed
down the proteolysis, the ripening indices for mould-ripened Civil cheese were lower especially at the rst
90 days of ripening. Inoculation of P. roqueforti in the
manufacture of mould-ripened Civil cheese contributed to
the proteolysis and pH of the cheese, but these contributions were not expected due to the high salt, low ripening
temperature and addition of Lor (whey cheese) cheese
as ller material. When Lor was incorporated in the Civil
cheese, P. roqueforti grew quite slowly on the cheese surface and had the undesirable effect of delaying the ripening of the cheese. Use of P. roqueforti changed only total
bacterial counts, yeasts and moulds. Other microbiological
composition did not change by addition of P. roqueforti.
In conclusion, the use of a dened strain of P. roqueforti
as secondary culture (without addition of Lor) may be
recommendable for the production of mould-ripened Civil
cheese.
ACKNOWLEDGEMENTS
The authors are grateful to the Scientic and Technological
Research Council of Turkey (TUBITAK, Turkey) for supporting this study (TOVAG-Project number 108 O 509). The
authors would also like to thank Leben Food and Dairy
Products (Erzurum, Turkey) for allowing the use of their
equipment and other facilities during the manufacture of
cheese.

602

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