Changes in Terpene Compo

Chapter 7
From Pasture to Cheese: Changes in Terpene

Composition
1,
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Publication Date: August 9, 2007 | doi: 10.1021/bk-2007-0971.ch007
Hedwig Schlichtherle-Cerny *, Miroslava I. Imhof ,

Estrella Fernndez Garca , and Jacques O. Bosset
1
Agroscope Liebefeld-Posieux (ALP), Swiss Federal Research Station

for Animal Production and Dairy Products, Berne, Switzerland
Instituto Nacional de Investigacin y Tecnologa Agraria y Alimentaria
(INIA), Madrid, Spain
The monoterpene composition of fresh grass originating from

altitudes between 1400 and 1920 m in Switzerland, of milk
stemming from the same sites, and of a Raclette-type cheese
which had been produced from the same milk were analyzed
by dynamic headspace-GC-MS. The milk and cheese samples
revealed additional monoterpenoids which had not been
detected in the grass, such as 3,7-dimethyl-1,6-octadiene, 3,7dimethyl-2-octene, 2,6-dimethyl-2,6-octadiene and menth-1ene. To study the impact of rumen fermentation on the
monoterpenes, a model plant, cow parsnip (Heracleum
sphondylium L.), was incubated with rumen fluid for 24 h. AH
terpenes were partially degraded, P-myrcene, (E)- and (Z)-ocimene almost completely. The degradation of the terpenes
was correlated with the formation of the additional new
compounds detected in milk and cheese. These compounds
may be formed by hydrognation during rumen fermentation.
2007 American Chemical Society
In Flavor of Dairy Products; Cadwallader, K., et al.;

ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
123
124

Introduction
Terpenes are secondary natural plant metabolites. Many of them are
bioactive constituents of medicinal herbs, some show antimicrobial activity
(1,2). Many terpenes exhibit characteristic odors and tastes, e.g. in essential oils,
fruits, vegetables and culinary herbs. In milk and cheese, however, terpenes have
not been described among the character impact compounds. Table I lists the
important aroma-active compounds of Swiss Gruyre cheese, a hard cheese
which is highly appreciated in Europe. According to Rychlik and Bosset (3,4)
the most potent odorants of Gruyere cheese, which are characterized by high
odor activity values, are sulfur-containing components derived from the amino
acid metabolism of the ripening cultures, such as methanethiol, methional and
dimethyltrisulfide, as well as phenylacetic acid and 2- and 3-methylbutanal.
However, terpenes are useful markers to determine the origin of dairy
products such as milk and cheese, i.e. to discriminate between highland and
lowland produce. The terpene profile proves a useful authenticity marker for
products with a protected designation of origin, so-called PDO products as
described by Zeppa and co-workers (5), Buchin and co-workers (6), and very
recently by Cornu and co-workers (7).
Mariaca and co-workers (8) investigated the volatile mono- and
sesquiterpenoids in different highland and lowland plant species. They could
show that highland pastures show a wide variety of different plant species. In
particular dicotyledonous plants usually rich in terpenoids are more abundant in
highland compared to lowland pastures. The latter are often composed of plant
species containing less dicotyledons and thus, are poor in terpenes. Bosset and
co-workers (9) compared hard cheese produced in the highlands to hard cheese
of the lowlands and found statistically significant differences in the chemical
composition such as terpenes, fatty acids and triglycerides between the highland
and lowland cheeses.
Only little, however, is known about the influence of rumen fermentation on
the monoterpenes present in plants and the changes they undergo from pasture to
cheese. The objective of the present study was to trace the monoterpene
composition of fresh grass, milk and a Raclette-type cheese produced from the
milk using dynamic headspace (purge + trap, P+T) gas chromatography in
combination with mass spectrometry (GC-MS).
To our knowledge this is the first study to investigate the influence of rumen
fermentation on the degradation of monoterpenes present in a model plant
commonly found on pastures. The results of the in vitro study will be compared
to the volatiles found in milk and in cheese.
A chiral stationary phase was employed to study whether the enantiomeric
ratios of the monoterpenes found in pasture are maintained in milk and in cheese,
and to investigate the impact of the bovine rumen microflora on the terpene

125
Table I. Aroma compounds of a lowland Gruyere cheese

Compound
Odor descriptor
Sulftirous
Methanethiol
Methional
Boiled potato-like
Dimethyltrisulfide
Cabbage-like
Phenylacetic acid
Honey-like
2-Methylbutanal
Malty
3-Methylbutanal
Malty
2-/3-Methylbutanoic acids
Sweaty
Acetic acid
Vinegar-like, pungent
Butanoic acid
Sweaty
5-Decalactone
Swet, coconut-like
Propanoic acid
Fruity, pungent
Phenylacetaldehyde
Honey-like
2,3-Diethyl-5-methylpyrazine Earthy
Methylpropanoic acid
Sweaty, rancid
()-2-Nonenal
Green
2-Ethyl-3,5-dimethylpyrazine Earthy
Hexanoic acid
Goat-like
Data are from references 3 and 4.
Cnc
700
99
136
7270
255
219
81000
298000
97000
1690
87000
20
0.6
41000
323
0.7
28000
activity
value
11300
495
54
39
26
17
8.1
5.5
5.4
4.2
2.9
1
1
0.5
0.4
0.3
0.3
composition of cow parsnip (Heracleum sphondylium L.), which was selected as

model plant.
Experimental
Materials
The fresh grass samples originated from six alps in the Canton of Vaud,
Switzerland, near the villages of Les Moulins and L'Etivaz, at altitudes from
1400 to 1920 m. They were collected at the end of August 2003. Seven alps
located around Les Moulins (Canton of Vaud, Switzerland, altitudes of the alps:
1400 to 1900 m) provided the cow's milk (one sample each), and one Raclettetype cheese investigated in the present study had been produced from the same
milk. Cow parsnip (Heracleum sphondylium L.) was collected twice, in midAugust 2003 near Schwarzenburg (Canton of Berne, Switzerland, altitude
around 700 m) and at the end of August 2003 in La Fretaz near St. Croix in the
Swiss Jura (Canton of Vaud, Switzerland, altitude around 1200 m). The grass,
the milk and the cow parsnip samples were pooled to form composite samples.
The samples were kept frozen at -20 C before use.


126
Rumen fluid was collectedfrom3 healthy cows aged between 5 and 8 years
with an artificial fistula connected to the rumen. The cows had been fed with hay
ad libitum for two weeks prior to the study.
The composite grass sample (5 g) was roughly chopped and mixed with
MilliQ water (100 mL) in a Polytron PT 3000 mixer (Kinematica, Littau,
Switzerland) at 10000 rpm for 2 min. The suspension (2 mL) was diluted with
MilliQ water (100 mL) and 2 mL of the diluted suspension were pipetted into a
25 mL non-fritted sparger (Schmidlin, Neuheim, Switzerland) for the P+T
analysis. Cow parsnip (2 g) was ground and suspended in phosphate buffer pH
6.6 (30 mL) at the same pH as the rumen fluid according to (10). Rumen fluid
(15 mL) was added and the suspension incubated at 39 C for 24 h in a shaking
mixer under argon atmosphere to prevent oxidation. The suspension (1 mL) was
diluted with MilliQ water (100 mL) and 2 mL of the dilution were employed for
the P+T analysis.
Milk (20 mL) was directly pipetted into the sparger for subsequent P+T
analysis. The Raclette-type cheese was finely grated and 5 g were weighed into a
beaker, mixed with MilliQ water (45 mL) and homogenized at 10000 rpm
(Polytron) for 2 min. Immediately after homogenization the sample (2 g) was
weighed into a sparger.
Instrumental Analysis
Dynamic headspace extraction (Purge + trap, P+T) using a LSC 2000 P+T
system equipped with a cryofocusing unit and a trap no. 8 (Tekmar, Cincinnati,
OH) was employed for the extraction of the monoterpenoids according to an
adapted method from Mariaca and co-workers (8). The samples were weighed
into a 25 mL non-fritted sparger (Schmidlin, Neuheim, Switzerland) and
extracted at 40 C using a water bath. The P+T conditions were as follows:
nitrogen was used as purge gas at 41 mL/min purge flow (vent); purge 20 min;
dry purge 11 min; cap cool down -150 C; desorb 4 min at 240C; inject 1.5 min
at 215 C; bake 10 min at 260 C; transfer line to GC 150 C.
Gas chromatography coupled to mass spectrometry (GC-MS) for the
separation and analysis of the monoterpenes was conducted on an Agilent 5890
Series II instrument (Palo Alto, CA) equipped with a 5971A MSD and a chiral
capillary column CP-Chirasil-Dex CB (25 m x 0.25 mm x 0.25 |im film
thickness; Stehelin, Basel, Switzerland) using helium as carrier gas at 50 kPa
(1.2 mL/min) at 35 C. The temperature program ran from 35 C (0.5 min hold)
to 215 C at a linear gradient of 2.5 C/min. The temperature of the GC-MS
transfer line was set to 280 C, the MSD scanned in the electron impact (EI)
mode at 70 eV from m/z 26 to 350 at 0.8 scans/s. Terpenes were identified by
their identical linear GC retention indices and mass spectra with the ones of


127
reference terpenes (Fluka, Buchs, Switzerland). The terpene metabolites were
tentatively identified based upon comparison of their mass spectra with
commercial mass spectrometry databases and retention indices in the literature.
Single ion monitoring (SIM) of the ions m/z 93, 95, 119, 70, or 69 for the
monoterpenes and the terpene metabolites, respectively, was employed for
semiquantitation comparing their peak heights before and after incubation with
rumen fluid. The ions m/z 136 and 93 were selected as qualifier ions for the
monoterpenes and their metabolites, respectively. The samples were analyzed
once. The software SPSS version 11 (SPSS Inc., Chicago, IL) was used for
correlating the degradation of (-)-p-ocimene, 8-3-carene and P-myrcene and
the formation of menth-l-ene, 3,7-dimethyl-l,6-octadiene and 3,7-dimethyl-2octene. Except for the correlation the data were not statistically treated.
Results and Discussion
The grass sample consisted of plants of various families as described in (6),

i.e. Apiaceae, Asteraceae, Campanulaceae, Rosaceae, Fabaceae, Geraniac
Lamiaceae, Plantaginaceae, Ranunculaceae, Polygonaceae, Rubiaceae,
also Poaceae, the family to which common grass belongs. The term "grass" will
be used for the mixture of the mentioned plant species. The monoterpene
composition of fresh grass was analyzed by dynamic headspace gas
chromatography-mass spectrometry using a chiral stationary phase. P-Myrcene,
p-cymene, the two enantiomers (+)- and (-)-limonene, (-)-P-ocimene and (Z)P-ocimene, y-terpinene and (-)-ot-pinene were the predominant monoterpenes in
the grass samples analyzed (data not shown). Figure 1 depicts the structures of
the monoterpenes detected in the present study. Sabinene, cc-thujene, (+)-apinene, a-phellandrene, the two enantiomers (-)-P-pinene and (+)-P-pinene, and
oc-terpinolene were only minor components. Table II compares the
monoterpenoids found infreshgrass, in milk, and in the Raclette-type cheese.
In the milk samples collected from farms from the same sites the additional
monoterpenes a-terpinene, 8-3-carene as well as a-phellandrene and
(-)-camphene were detected as minor constituents, however, no ()-P-ocimene
was found. Interestingly, the analysis revealed further monoterpenoids
tentatively identified as 3,7-dimethyl-l,6-octadiene, 2,6-dimethyl-2,6-octadiene,
3,7-dimethyl-2-octene, and camphane. These monoterpenoids had been absent in
fresh grass. In the Raclette-type cheese produced from the milk basically the
same monoterpenes were found as in milk, except for 8-3-carene. Additionally,
2-carene, ()-P-ocimene and the tentatively identified menth-l-ene were
detected. 3,7-Dimethyl-l,6-octadiene, 2,6-dimethyl-2,6-octadiene, 3,7-dimethyl2-octene, camphane and menth-l-ene were only detected in the milk and cheese


2-Carene
5-3-Carene
a-Pinene
Limonene
Sabinene
p-Pheilandrene
Camphane
p-Pinene
a-Terpinene (Z)-p-Ocimene
3,7-Dimethyl- p-Menth-1-ene 3,7-Dimethyl- 2,6-Dimethyl1,6-octadiene

2-octene
2,6-octadiene
a-Phellandrene Camphene
p-Myrcene
y-Terpinene
()-p-Ocimene
00
Figure 1. Structures of monoterpenes and monoterpene metabolites tentatively identified in grass,

milk, Raclette-type cheese and in cow parsnip
a-Terpinolene
p-Cymene
a-Thujene

129
Table II. Monoterpenoids detected in grass, milk and Raclette-type cheese

Compound
cc-Thujene
P-Myrcene
2-Carene
(-)-a-Pinene
Sabinene
(+)-a-Pinene
a-Terpinene
(Z)-P-Ocimene
(+)-P-Pinene
()-P-Ocimene
5-3-Carene
p-Cymene
a-Phellandrene
(-)-Camphene
(+)-Camphene
(-)-Limonene
(+)-Limonene
(-)-P-Pinene
p-Phellandrene
y-Terpinene
a-Terpinolene
3,7-Dimethyl-1,6-octadiene
Menth-l-ene
3,7-Dimethyl-2-octene
Camphane
Grass
Milk
Raclette-type
cheese
s
-
s
s
s
s
-
s
-
samples, however not in fresh grass. The presence of these compounds had
already been described in alpine milk by Buchin and co-workers (6).
The enantiomeric ratios of selected monoterpenes were analyzed to examine
whether the ratios changed from pasture to cheese, but no consistent trend could
be observed. In the grass sample (-)-a-pinene was prevalent over the
(+)-enantiomer, and (+)-limonene over (-)-limonene. In contrast, in milk and in
cheese (+)-oc-pinene predominated. As already found in the grass sample,
(+)-limonene prevailed also in milk and in the cheese samples over
(-)-limonene. Presumably, the cows had grazed on a pasture with plants
producing more (+)-ct-pinene.

130

Incubation of cow parsnip with rumen fluid

The exclusive presence of the dimethyl octadienes, menth-l-ene and the
other tentatively identified compounds in milk and cheese raised the hypothesis
that these compounds might have been formed during rumen fermentation. Many
of the monoterpenes shown in Figure 1 show two or three double bonds, whereas
3,7-dimethyl-l,6-octadiene, 2,6-dimethyl-2,6-octadiene, 3,7-dimethyl-2-octene,
camphane and menth-l-ene have two or only one double bond. Hydrogenation
during rumen fermentation might be involved in their formation. The rumen flora
is known to be strictly anaerobic. It produces hydrogen among other gases and
biohydrogenation reactions convert e.g. unsaturated fatty acids into saturated
fatty acids (77). Cow parsnip, Heracleum sphondylium L., was selected as a
model to investigate a potential correlation between the degradation of certain
monoterpenes and the formation of the above-mentioned compounds. Cow
parsnip belongs to the family Apiaceae, is found abundantly in pastures and rich
in terpenoids as described by Mariaca and co-workers (8).
Ground cow parsnip was incubated with rumen fluid over 24 h and monitor
the changes in its monoterpene composition. After 12 h of fermentation all
monoterpenes were at least partially degraded. The rate and extent of
degradation varied for the different compounds. Figure 2 illustrates the
degradation of selected monoterpenes after 12 h of incubation.
(Z)-(J-ocimene, ()-P-ocimene, a-phellandrene and P-myrcene were
completely or almost completely degraded, while around 40 % of (+)-limonene
and p-cymene, and 20 to 35 % of most other monoterpenes remained after 12 h
of incubation.
During incubation with rumen fluid the formation of the monoterpenoid
compounds that had been detected in milk and in cheese was observed. The
extent and rate of formation of these compounds varied, as it could be seen for
the degradation of the different monoterpenes.
Statistically significant (p < 0.05) negative correlations were found for the
degradation of ()-p-ocimene and the co-eluting 8-3-carene and the p-myrcene
and the formation of menth-l-ene, 3,7-dimethyl-l,6-octadiene and 3,7-dimethyl2-octene. ()-P-Ocimene, 8-3-carene and P-myrcene were almost completely
and rapidly degraded during the first 8 h of incubation. Menth-l-ene increased
steadily during the first 8 h of incubation, then was slowly degraded from 8 to
12 h of rumen fermentation. After 12 h the menth-l-ene signal intensity dropped
rapidly over the next 4 h as shown in Figure 3.
The formation of 3,7-dimethyl-l,6-octadiene began after 2 h of rumen
fermentation and reached its maximum after 8 h. Then it degraded slightly. At
first in parallel to 3,7-dimethyl-l,6-octadiene, but after 4 h of incubation only,
the onset of 3,7-dimethyl-2-octene could be observed. The peak signal increased
rapidly after 12 h of rumen fermentation.

131
H Before incubation
After 12 h incubation
700
600
' 500
"(/)
c
&>
a 400
200

100
1
1 il,
llI ll
Figure 2. Monoterpenoids of cow parsnip before and after 12 h of incubation

with rumen fluid
The formation of the newly formed compounds may be explained by

hydrogenation reactions by the rumen flora or by enzymes present in the rumen
fluid as schematically presented in Figure 2. Hydrogenation of the double bond
at position 3 of P-myrcene or of (Z)- and ()-P-ocimene yields 3,7-dimethyl-l,6octadiene, a shift of the double bond at position 1 might explain the formation of
2,6-dimethyl-2,6-octadiene which had also been detected in the milk and
Raclette-type cheese samples. A further hydrogenation of 2,6-dimethyl-2,6octadiene yields 3,7-dimethyl-2-octene.
Conclusions
The monoterpene composition offreshgrass, milk, and cheese was analyzed
by dynamic headspace GC-MS. It was shown that milk and cheese revealed
additional monoterpenes which had not been detected in the grass samples. A
model plant, cow parsnip, Heracleum sphondylium L., was incubated in vitro
with rumen fluid to investigate for the first time the relationship between the
degradation of monoterpenes and the formation of these additional
monoterpenoids. The monoterpenes of cow parsnip were at least partially
degraded during incubation with rumen fluid and 3,7-dimethyl-l,6-octadiene,
3,7-dimethyl-2-octene, 2,6-dimethyl-2,6-octadiene together with camphane and

(ZT)-B-Ocimene
8-3-carene
B-Myrcene
Menth-l-ene

3,7-Dimethyl-2-octene
12
16
24
Incubation time (h)
Figure 3. Degradation and new formation ofselected monoterpenoids du

incubation of Heracleum sphondylium L.with rumen fluid.
Hydrogenation
by rumen flora
JL
t-Myrcene
(E)-p-Ocimene (Z)-p-Ocimene
3,7-Dimethyl1,6-octadiene
2,6-Dimethyl2,6-octadiene
^Further
hydrogenation
3,7-Dimethyl2-octene
Figure 4. Hypothetic schematic formation of selected monoterpenoids

by hydrogenation

133
menth-l-ene were formed. It is hypothesized that p-myrcene, (E)- and (Z)-Pocimene are hydrogenated during incubation with rumen fluid to yield these
components. The results show that monoterpenes are partially degraded during
rumen fermentation and new compounds are formed, which are also transferred
into milk and cheese.

Acknowledgments
The authors thank Roland Gauch and Bernard Jeangros for their help
collecting the samples, Kurt Zbinden for providing the rumen fluid and Frigga
Dohme for useful discussions.
References
1.
Nostro, A.; German, M.P.; D'Angelo, V.; Marino, A.; Cannatelli, M.A.
Lett. Appl.Microbiol.2000, 30, 379-384.
2. Delgado, B.; Palop, A.; Fernndez, P.S.; Periamo, P.M. Eur. Food Res.
Technol 2004,275, 188-193.
3. Rychlik, M.; Bosset, J. O. Int. Dairy J. 2001, 11, 895-901.
4. Rychlik, M.; Bosset, J.O. Int. Dairy J. 2001, 11, 903-910.
5. Zeppa, G.; Giordano, M. In Abstracts of the 5th International Meeting on
Mountain Cheese, INRA, Ed., Ar&ches, France, 2003
6. Buchin, S.; Salmon, J.C.; Carnat, A.P.; Berger, T.; Bugaud, C.; Bosset, J.O.
Mitt. Lebensm. Hyg. 2002, 93, 199-216.
7. Cornu, A.; Kondjoyan, N . ; Martin, B.; Verdier-Metz, I.; Pradel, P.;
Berdagu, J.L.; Coulon, J.B. J. Sci. Food Agric. 2005, 85, 2040-2046.
8. Mariaca R.; Berger T.; Gauch R.; Imhof M., Jeangros B.; Bosset J.O. J.
Agric. FoodChem. 1997, 45, 4423-4434.
9. Bosset J.O.; Berger T.; Btikofer U.; Collomb M.; Gauch R.; Lavanchy P.;
Sieber R. Agrarforschung 1998, 5, 363-366.
10. Fievez, V.; Dohme F.; Danneels, M.; Raes K.; Demeyer, D. Anim. Feed Sci.
Technol. 2003, 704,41-58.
11. Collomb M.; Sieber R.; Btikofer U. Lipids 2004, 39, 355-364.


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Chapter 7

From Pasture to Cheese: Changes in Terpene

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

Hedwig Schlichtherle-Cerny *, Miroslava I. Imhof ,

Agroscope Liebefeld-Posieux (ALP), Swiss Federal Research Station

The monoterpene composition of fresh grass originating from

2007 American Chemical Society

In Flavor of Dairy Products; Cadwallader, K., et al.;

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

In Flavor of Dairy Products; Cadwallader, K., et al.;

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

composition of cow parsnip (Heracleum sphondylium L.), which was selected as

In Flavor of Dairy Products; Cadwallader, K., et al.;

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

In Flavor of Dairy Products; Cadwallader, K., et al.;

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

Results and Discussion

The grass sample consisted of plants of various families as described in (6),

In Flavor of Dairy Products; Cadwallader, K., et al.;

In Flavor of Dairy Products; Cadwallader, K., et al.;

3,7-Dimethyl- p-Menth-1-ene 3,7-Dimethyl- 2,6-Dimethyl1,6-octadiene

Figure 1. Structures of monoterpenes and monoterpene metabolites tentatively identified in grass,

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In Flavor of Dairy Products; Cadwallader, K., et al.;

Downloaded by STANFORD UNIV GREEN LIBR on May 10, 2012 | http://pubs.acs.org

Incubation of cow parsnip with rumen fluid

In Flavor of Dairy Products; Cadwallader, K., et al.;

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Figure 2. Monoterpenoids of cow parsnip before and after 12 h of incubation

The formation of the newly formed compounds may be explained by

In Flavor of Dairy Products; Cadwallader, K., et al.;

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Incubation time (h)

Figure 3. Degradation and new formation ofselected monoterpenoids du

Figure 4. Hypothetic schematic formation of selected monoterpenoids

In Flavor of Dairy Products; Cadwallader, K., et al.;

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In Flavor of Dairy Products; Cadwallader, K., et al.;

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