ARTICLE IN PRESS

LWT 40 (2007) 11401145

www.elsevier.com/locate/lwt

The potential for carbon dioxide formation during ripening

of acid curd cheese
Doris Jaros, Thea Hofmann, Harald Rohm
Institute of Food Technology and Bioprocess Engineering, Technische Universitat Dresden, D-01062 Dresden, Germany
Received 7 April 2006; received in revised form 28 June 2006; accepted 4 July 2006

Abstract
Inspired by recent reports on high concentrations of carbon dioxide in the atmosphere of ripening chambers used in acid curd cheese
production, small-scale experiments were performed to systematically investigate sources of CO2 formation. In a closed system with a
ratio of cheese mass to air volume close to that in industrial scale, up to approximately 16% (v/v) CO2 were observed within 3 d of
maturation at 24 1C. Without addition of ripening salts (CaCO3, NaHCO3) the initial carbon dioxide formation was delayed, but
maximum CO2 levels were still much higher than admissible workplace concentrations. Control experiments with quarg, which was
pasteurized for yeast inactivation, revealed that growth and activity of yeasts has to be considered as the most important source for
carbon dioxide formation. The results of the study strongly point on the necessity of preventive measures for ensuring the safety of
employees.
r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Acid curd cheese; Ripening; Carbon dioxide formation; Yeasts

1. Introduction
Acid curd cheeses are traditional cheeses with a restricted
regional distribution and comprise, among others, varieties
such as Harzer Kase or Olmutzer Quargel. The technology
of acid curd cheese manufacture has been summarized
recently (Bockelmann et al., 2002; Bockelmann, Willems,
Neve, & Heller, 2005). In principle, a crumbly quarg with a
dry matter of approximately 320 g kg1 is produced by
acidifying heat-treated skim milk with thermophilic yogurt
starters. Immediately after production the quarg contains
approximately 104105 cfu g1 yeasts (Bockelmann et al.,
2002) which are either contaminants, or which originate
from an inoculate added to the milk prior to fermentation.
Preripening during storage and transport of the quarg to
the cheese-making companies result in a development of
the viable yeast counts to, typically, 106107 cfu g1. After
mixing the quarg with NaCl, ripening salts (usually a
Corresponding author. Tel.: +49 351 463 34985;
fax: +49 351 463 37126.
E-mail address: doris.jaros@tu-dresden.de (D. Jaros).

mixture of CaCO3 and NaHCO3; Kammerlehner, 2003),

spices (e.g., black cumin), a small portion of grinded
mature cheese which serves as inoculant, and some water
for adjusting dry matter to approximately 280 g kg1, small
cheese wheels of approximately 40 g are formed. Subsequent maturation comprises two steps: (1) At elevated
temperature, e.g., 30 1C for 1 d (Bockelmann et al., 2005) or
24 1C for 23 d (Kessler, 2002), the cheese mass is
deacidied through the action of the ripening salts and
the growth of the yeasts, mainly Kluyveromyces marxianus
and Candida krusei; (2) after spraying with smear bacteria,
the cheeses undergo signicant changes at approximately
1416 1C within a few days, resulting in the formation of
typical color and avor and in texture development
through the ongoing metabolic activities of surface
microorganisms (Eliskases-Lechner & Ginzinger, 1995;
Leclercq-Perlat, Oumer, Bergere, Spinnler, & Corrieu,
2000; Leclercq-Perlat, Oumer, Buono et al., 2000).
Additionally, further ripening occurs during cold storage.
According to current regulations for occupational safety
in Germany the admissible workplace concentration for
CO2 in air is 5000 ppm or 0.5% (v/v); this value might be

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2006.07.002

ARTICLE IN PRESS
D. Jaros et al. / LWT 40 (2007) 11401145

exceeded by a factor of 2 for two 15 min periods during one

working shift (Anonymous, 2006). In several measurements, the Berufsgenossenschaft Nahrungsmittel und
Gaststatten, the German institution for statutory accident
insurance and prevention in the foodstuffs industry and the
catering trade, observed exceeded carbon dioxide concentrations in the atmosphere of ripening rooms, being
potentially hazardous if these are entered for control
purposes (Herzberg, 2006). During processing and ripening
of acid curd cheese several sources of carbon dioxide must
be taken into account (Beresford, Fitzsimons, Brennan, &
Cogan, 2001; Brennan, Cogan, Loessner, & Scherer, 2004;
Kammerlehner, 2003): (1) CaCO3 and NaHCO3 liberate
CO2 in an acidic milieu, causing an increase of pH in the
cheese base and a volume expansion of the cheese mass.
This process starts immediately after addition until the
salts are consumed; (2) the oxidation of lactic acid by
yeasts leads to the formation of pyruvate which, when
enzymatically decarboxylated, results in the formation of
acetaldehyde and CO2; (3) depending on the pH of the
system free amino acids, liberated during proteolysis, may
be decarboxylated to amines or oxidized to acetaldehyde,
also contributing to CO2 formation. In the early stages of
acid curd cheese maturation the reactions (1) and (2) may
be considered as the predominant sources of carbon
dioxide.
To the best of our knowledge, there is only one recent
publication dealing with potentially hazardous atmospheric conditions in cheese ripening rooms. Haas and
Spillmann (2001) monitored ammonia and CO2 in 67 Swiss
ripening rooms, presumably for hard and semi-hard
cheeses, and found an average CO2 concentration of
0.37% (v/v) with a maximum value of 0.82% (v/v). The
purpose of our study was to monitor CO2 formation during
acid curd cheese ripening in small scale, and to quantify the
potential and specic contribution of the above-mentioned
factors.
2. Materials and methods
2.1. Materials
All chemicals were analytical grade (Merck GmbH,
Darmstadt, Germany). Several batches of quarg (dry
matter content: approximately 320 g kg1) were purchased
from a small cheese-making company (Kaserei Klinnert &
Sohn, Rabenau/Karsdorf, Germany). Each batch was split
into 1 kg aliquots, which were packed in polythene bags
and deep-frozen until used. Acid curd cheese purchased in
a local supermarket served as inoculation material.
2.2. Small-scale cheese manufacture
Prior to each experiment, one package of quarg was
thawed in a refrigerator overnight. A dispersion of various
additives (CaCO3, NaHCO3, NaCl, and nely grated
mature cheese) in distilled water was added to the quarg

1141

to ensure a dry matter content of 290300 g kg1 in the

nal cheese base. The amount of the additives in the
dispersion also depended on quarg dry matter and, for
cheese serving as reference, was adjusted to achieve a
concentration in the cheese base of 10, 10, 20, and
40 g kg1, respectively. The addition of mature cheese,
usually denoted as Culture cheese (Bockelmann et al.,
2005), is equivalent to the oldyoung smearing method
used in the production of smear-ripened cheeses and still
applied in industrial manufacture of acid curd cheese.
Apart from NaCl, the one or other compound was
systematically withdrawn from the recipe to study its
effects on CO2 formation. After thoroughly homogenizing
the mixture with an N50 mixer equipped with a at B
beater (Hobart GmbH, Offenburg, Germany), small cheese
wheels (diameter, 45 mm; height, 20 mm; mass, approximately 40 g) were formed by means of perspex rings.
The cheese wheels (24 pieces per production) were then
placed on 3 perforated stainless steel sheets and transferred
into a double-walled perspex chamber with a volume of
approximately 14 l (inner dimensions: width, 170 mm;
height, 200 mm; depth, 400 mm). The ratio of cheese mass
to chamber volume is close to the value used in industrial
acid curd cheese ripening (e.g., 14 t in 180 m3). Temperature
(T, 1C) in the chamber was kept constant by means of a
Peltier element, which was powered by a self-programmed
control unit. A small computer fan, operated at approximately 200 rpm, was responsible for air circulation within
the chamber. Air from the chamber was sucked into a
Binos 1000 infrared continuous gas analyser (FisherRosemount GmbH & Co., WeXling, Germany) with a ow
rate of 0.5 l min1 and, after passing the CO2 measuring
cell, pumped back into the chamber. Prior to the
measurements the system was calibrated according to the
instructions of the manufacturer. A relative humidity (r.h.,
%) of 494% was achieved by placing approximately 0.5 l
water in an aluminum dish, which covered the bottom of
the chamber. T and CO2 values were continuously logged
in intervals of 2 s and 2 min, respectively. After adjusting T
to 2470.2 1C for 3 d in the rst maturation period, the
chamber was opened and the cheeses were smeared with a
suspension containing 0.5% (v/v) DH1 yeast starter
(Debaryomyces hansenii) and 0.5% (v/v) RK starter
(Brevibacterium aurantiacum; Bundesanstalt fur Alpenlandische Milchwirtschaft, Rotholz, Austria) in a sterile
40 g l1 aqueous NaCl solution. In the second maturation
step, T was set to 1870.2 1C for 5 d.
2.3. Analytical methods
For all measurements on acid curd cheese, 3 randomly
selected cheese wheels were carefully homogenized, and a
representative aliquot was taken for analysis. Moisture
content of quarq, cheese base and acid curd cheese was
determined by the oven method at 102 1C (IDF, 1982), and
pH of grated cheese was measured with a pin-shaped
electrode. Total nitrogen (N) content and the N fraction

ARTICLE IN PRESS
1142

D. Jaros et al. / LWT 40 (2007) 11401145

soluble in 120 g l1 trichloracetic acid (TCA) were determined by the Kjeldahl method (IDF, 2001). Viable yeast
counts were obtained after homogenizing 5 g cheese in
45 mL sterile 9.5 g l1 NaCl-solution and plating decimal
solutions thereof on yeast extract glucose chloramphenicol
agar (incubation at 25 1C for 5 d).
3. Results and discussion
3.1. Characterization of carbon dioxide formation during
cheese ripening
Preliminary experiments were performed to characterize
the tightness of the ripening chamber. Without loading
cheeses a CO2-enriched atmosphere was created by
dissolving different amounts of 1:1-mixtures of CaCO3
and NaHCO3 in 0.25 l 2% lactic acid. Fig 1 shows that,
depending on the amount of salts used for carbon dioxide
generation, the CO2 concentration reached a specic
maximum within 1 h and then decayed continuously within
a time frame of 24 h at T 24 1C. There are two
reasonable explanations for the decrease in concentration:
(1) The chamber is not completely air-tight and has
leakages, especially along the sealing at the edges or along
the cable outlets; (2) losses do also originate from the
continuous circulation through the measuring unit and the
accompanying exible tube system. Independent of the
initial CO2 concentration, however, the decay is calculable
as it followed a rst-order reaction with a rate constant of
approximately 0.037 h1. These unavoidable losses have to
be considered in the further interpretation of our data.
Fig. 2a depicts the development of carbon dioxide in the
ripening chamber for 3 replicate productions of reference
cheeses made from quarg batch #1. The concentration
increased to approximately 2% (v/v) within 3 h after

Fig. 1. Loss of carbon dioxide from the maturation chamber. CO2 was
generated by dissolving different amounts of ripening salts in 2% lactic
acid. Air temperature was set to 24 1C. Equations refer to CO2 decay
models.

Fig. 2. Development of carbon dioxide during ripening of acid curd

cheese: (a) CO2 prole of 3 replicate productions made from quarg batch
#1. Inset: rst derivative of the CO2time curve in the initial stage of
maturation; and (b) averaged CO2 proles from replicate productions
made from quarg batch #1 (J, n 3), batch #2 (K, n 2), batch #3 (&,
n 2) or batch #4 (, n 2). Bar height refers to the averaged variation
range. Maturation conditions: 0otimeo3 d, 24 1C at 494% r.h.;
3otimeo7 d, 18 1C at 494% r.h.

starting the experiment, indicating that CO2, which was

not released during cheese base mixing, is now liberated
from the ripening salts. Subsequently, the slope of the
concentration curve (inset to Fig. 2a) diminishes and
reaches a local minimum within a time frame of 69 h;
afterwards, the CO2 formation rate increases again. The
maximum CO2 concentration of approximately 10% (v/v)
was measured after 12 d of maturation.
It can be seen from Fig. 2b that the CO2 concentration in
the ripening chamber does largely depend on the base
quarg. In case of quarg #1 showing a pH of 3.92 and a
yeast count of 1.1  106 cfu g1 the addition of
culture cheese increased the yeast count to 2.6  106 cfu g1
(Table 1). During maturation the yeast count increased to
7.8  106 cfu g1 (3 d) and, furthermore, to 8.5  106 cfu g1
(7 d). The CO2 proles from acid curd cheese produced
from quarg batches #2 and #3 are similar in shape to that
of batch #1 but show signicantly higher maximum
concentrations which, as can be seen from Table 1, are
interrelated with the increase in the viable yeast count in
the rst stage of maturation. The cheese base prepared
from quarg #4 showed a higher yeast count than the other
samples (4.7  106 cfu g1); hence, we observed a more
pronounced CO2 increase at the beginning of the ripening,
up to approximately 12 h. In all experiments, the CO2
concentration reached a maximum after approximately
1.5 d of ripening. The subsequent CO2 decrease appears to
be a consequence of the leakages mentioned above.

ARTICLE IN PRESS
D. Jaros et al. / LWT 40 (2007) 11401145

1143

Table 1
Development of viable yeast counts and pH during maturation of acid curd cheese
Quarq batch

#1
#2
#3
#4

n 3
n 2
n 2
n 2

Viable yeast counts (cfu g1) in

pH () in
Quarg

Acid curd
cheese (3 d)

Acid curd
cheese (7 d)

Quarg

Base
preparation

Acid curd
cheese (3 d)

Acid curd
cheese (7 d)

3.92
3.98
3.86
4.06

5.92
5.81
5.68
5.98

6.94
6.82
7.11
7.19

1.1  106
1.4  106
2.9  105
2.0  106

2.6  106
2.4  106
1.0  106
4.7  106

7.8  106
1.0  107
1.8  107
9.2  106

8.5  106
1.8  107
2.5  107
2.1  107

However, when comparing the kinetics of CO2 losses

(approximately 60% in 24 h; see Fig. 1) and the rate of CO2
decrease shown in Fig. 2b, it becomes obvious that there
must still be a sufcient amount of CO2 production in this
particular ripening period.
After smearing the surface of the cheeses made from
quarg samples #2, #3 and #4 with a mixture of D. hansenii
and B. aurantiacum, the CO2 concentration was further
monitored. In this second stage of maturation the
formation of CO2 was much less pronounced, resulting in
maximum levels of approximately 3% (v/v). The almost
constant concentration in this ripening period indicates
that there is a continuous formation of carbon dioxide,
counterbalancing the unavoidable losses due to the
chamber characteristics.
3.2. Contribution of ripening salts and cheese flora to carbon
dioxide formation
The inuence of the addition of ripening salts and
mature cheese on CO2 formation during the rst stage of
ripening is depicted in Fig. 3. As compared to the reference
cheese (cheese base made from quarg #3; see also Fig. 2b),
CO2 formation was signicantly reduced when preparing a
cheese base either without CaCO3 or NaHCO3, or without
both salts. A detailed inspection of the concentrationtime
proles leads to the conclusion that two factors must be
responsible for these differences. Six hours after transferring the cheeses into the chamber, the CO2 concentration
was proportional to the amount of ripening salts added to
the cheese base: we measured 2.3% (v/v) CO2 when the
cheeses contained 20 g kg1 ripening salts, approximately
1.2% (v/v) CO2 when the cheeses contained 10 g kg1
ripening salts (either CaCO3 or NaHCO3), and 0.3% (v/v)
CO2 when no salts were added to the base quarq. In cheeses
with a reduced amount of ripening salts the delay prior to
the second steep increase in CO2 concentration is
presumably because of a delayed growth of the yeasts
which, in turn, is caused by the lower pH of the cheese base
(Fig. 4). As regards maturation progress a low initial pH
and a reduced deacidication is obviously interrelated with
a delayed proteolysis. For all cheeses in this particular set
of samples the dry matter distribution was very narrow,
showing values of 29774.4, 30473.3, and 31172.6 g kg1
for the cheese base and cheeses aged for 3 and 7 d,

Fig. 3. Development of carbon dioxide as affected by cheese base

composition. Sample identiers: J, reference cheese base made from
quarg #3 (n 2); &, cheese base w/o CaCO3 n 2; W, cheese base w/o
NaHCO3 n 2; X, cheese base w/o CaCO3 and NaHCO3 n 2; ,
cheese base w/o culture cheese n 2; m, cheese base w/o CaCO3,
NaHCO3 and culture cheese n 2; ., cheese base from pasteurized
quarg w/o culture cheese n 2.

respectively. Regarding experiments made with addition of

ripening salts but without addition of culture cheese, there
was a delay in the CO2 formation as well. After the rst
stage of maturation, these cheeses showed a slightly lower
pH than the reference sample. pH and proteolysis
measurements made after 7 d of maturation revealed,
however, no signicant differences. When only incorporating quarg, water and NaCl into the cheese base a negligible
amount of CO2 was liberated within the rst day; the initial
pH was approximately 4.2 and increased to approximately
5 after 7 d of maturation, and proteolysis within 3 d was
not detectable. The delay in carbon dioxide formation was
more pronounced than in cheeses made without ripening
salts but with addition of culture cheese. The CO2
concentration reached within 3 d, however, clearly indicates that the yeast ora in the quarg is able to multiply at
24 1C. A distinct proof that ripening salts are only
responsible for the initial CO2 formation, whereas the
major contribution to the carbon dioxide level comes
from yeast metabolism, is also evident from Fig. 3.

ARTICLE IN PRESS
1144

D. Jaros et al. / LWT 40 (2007) 11401145

cheese base was o10, and o103 cfu g1 were found after
3 d.
Cross-sections of selected cheeses which were, after
ripening for 7 d, further stored at 5 1C for 10 d, are shown
in Fig 5. Reference cheeses and cheeses made without
CaCO3 were almost identical in their appearance and in
their organoleptical characteristics, and the same was true
for cheeses made without NaHCO3 (data not shown). The
cheeses made without both salts, or without both salts and
without addition of mature cheese still showed a large
quarg core.
4. Conclusions

Fig. 4. Development of pH and proteolysis during ripening of acid curd

cheese. TCAN/TN, ratio of nitrogen soluble in 12% TCA to total
nitrogen. Sample identiers: J, reference cheese base made from quarg #3
n 2; &, cheese base w/o CaCO3 n 2; W, cheese base w/o NaHCO3
n 2; X, cheese base w/o CaCO3 and NaHCO3 n 2; , cheese base
w/o culture cheese n 2; m, cheese base w/o CaCO3, NaHCO3 and
culture cheese n 2.

The results of the study demonstrate that there is an

enormous potential for carbon dioxide formation during
ripening of acid curd cheese. Under the conditions applied
in our experimental setup, a certain amount of CO2
originates from the ripening salts, primarily used for pH
adjustment. When comparing the stoichiometric amount of
carbon dioxide to be released from a dened mixture (e.g.,
the reaction of equimolar concentrations of NaHCO3 and
lactic acid leads to the formation of equimolar concentrations of sodium lactate, water and carbon dioxide) with the
actual concentration in the closed ripening system it
becomes evident that most of the CO2 is liberated into
the atmosphere during preparation of the cheese base. As
regards the environmental conditions within ripening
chambers the activity of the yeast ora appears to be
primarily responsible for CO2 formation. As a consequence
there is a denite need for technical and organizational
actions, which ensure the safety of employees. A continuous monitoring of the carbon dioxide concentration is
recommended, as are limit values to regulate access to the
ripening rooms. Currently, the applicability of using
breathing protection, which allows riskless entering despite
high CO2 concentrations, is under evaluation (Herzberg,
2006).
Acknowledgments

Fig. 5. Cross-sections of acid curd cheese ripened for 7 d: (a) reference

cheese; (b) cheese made from a preparation w/o CaCO3; (c) cheese made
from a preparation w/o CaCO3 and w/o NaHCO3; and (d) cheese made
from a preparation w/o CaCO3, w/o NaHCO3 and w/o culture cheese.

We pasteurized quarg by simply immersing a tightly sealed

polythene bag into a water bath at 65 1C for 4 h, and
prepared a cheese base by adding water, NaCl and ripening
salts. The CO2 concentration in the ripening chamber
reached a maximum of 3.4% (v/v) after approximately 8 h
and, subsequently, showed a continuous decay, as could be
expected when no additional carbon dioxide is produced
(see Fig. 1). The viable yeast counts in this particular

The research project was performed in cooperation with

the Berufsgenossenschaft Nahrungsmittel und Gaststatten,
Mannheim, Germany, and we thank Wolf-Dietrich Herzberg, Reinhard Jachmann and Karla Neubert (Geschaftsbereich Pravention, Dresden) for the fruitful discussions.
The technical assistance of Martin Mutterlein and Henriette
Groh is greatfully acknowledged.
References
Anonymous. (2006). Technische Regeln fur GefahrstoffeArbeitsplatzgrenzwerte (TRGS 900). Dortmund, Germany: Bundesanstalt fur
Arbeitsschutz und Arbeitsmedizin.
Beresford, T. P., Fitzsimons, N. A., Brennan, N. L., & Cogan, T. M.
(2001). Recent advances in cheese microbiology. International Dairy
Journal, 11, 259274.

ARTICLE IN PRESS
D. Jaros et al. / LWT 40 (2007) 11401145
Bockelmann, W., Willems, P., Jager, B., Hoppe-Seyler, T., Engel, K. J., &
Heller, K. J. (2002). Reifung von Harzer Kase. Kieler Milchwirtschaftliche Forschungsberichte, 54, 317335.
Bockelmann, W., Willems, P., Neve, H., & Heller, K. J. (2005). Cultures
for the ripening of smear cheeses. International Dairy Journal, 15,
719732.
Brennan, N. M., Cogan, T. M., Loessner, M., & Scherer, S.
(2004). Bacterial surface-ripened cheeses. In P. F. Fox, P. L. H.
McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), Cheese.
Chemistry, physics and microbiology (pp. 199226). London:
Elsevier.
Eliskases-Lechner, F., & Ginzinger, W. (1995). The yeast ora of surfaceripened cheeses. Milchwissenschaft, 50, 458462.
Haas, J., & Spillmann, H. (2001). Ammoniak in der Raumluft von
Kasekellern: Eine arbeitshygienische Abklarung. Milchwissenschaft,
56, 672675.
Herzberg, W.-D. (2006). Personal communication. Berufsgenossenschaft
Nahrungsmittel und Gaststatten, Mannheim, Germany.

1145

IDF. (1982). Cheese and processed cheesedetermination of total solids content.

IDF-Standard 4A. Brussels, Belgium: International Dairy Federation.
IDF. (2001). Milkdetermination of nitrogen contentKjeldahl method.
IDF-Standard 20-1. Brussels, Belgium: International Dairy Federation.
Kammerlehner, J. (2003). Kasetechnologie. Freising, Germany: Verlag
Freisinger Kunstlerpresse W. Bode.
Kessler, H. G. (2002). Food and bio process engineeringDairy technology.
Munchen, Germany: Verlag A. Kessler.
Leclercq-Perlat, M.-N., Oumer, A., Bergere, J.-L., Spinnler, H.-E., &
Corrieu, G. (2000). Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening ora in controlled production of smear
soft cheese from reconstituted milk: Growth and substrate consumption. Journal of Dairy Science, 83, 16651673.
Leclercq-Perlat, M.-N., Oumer, A., Buono, F., Bergere, J.-L., Spinnler,
H.-E., & Corrieu, G. (2000). Behavior of Brevibacterium linens and
Debaryomyces hansenii as ripening ora in controlled production of
smear soft cheese from reconstituted milk: Protein degradation.
Journal of Dairy Science, 83, 16741683.