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Chapter 7
FromPasturetoCheese:Changesin Terpene
Composition
Hedwig
Schlichtherle-Cerny
1,
*, Miroslava I.Imhof
1
,
EstrellaFernndez Garca
2
,andJacquesO.
Bosset
1
1
Agroscope Liebefeld-Posieux ALP),SwissFederalResearch Station
for Animal Production and Dairy
Products, Berne,
Switzerland
2
InstitutoNacional deInvestigacinyTecnologaAgraria y Alimentaria
INIA),
Madrid,
Spain
Themonoterpene 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-typecheese
whichhad been produced
from
the same milkwere analyzed
bydynamicheadspace-GC-MS.Themilkandcheesesamples
revealed additional monoterpenoids
which
had not been
detected in the grass, such as 3,7-dimethyl-1,6-octadiene, 3,7-
dimethyl-2-octene, 2,6-dimethyl-2,6-octadiene and menth-1-
ene. 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.
A H
terpeneswere 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 byhydrognationduring rumen fermentation.
2007
American
ChemicalSociety
123
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Introduction
Terpenes are secondary natural plant metabolites. Many of them are
bioactive constituents of medicinal herbs, some show antimicrobial activity
1,2).Manyterpenesexhibit characteristic odors and
tastes,
e.g. in essential
oils,
fruits, vegetables and
culinary
herbs. In
milk
and cheese, however,terpeneshave
not been described among the character impact compounds. Table I lists the
important aroma-active compounds of SwissGruyre cheese, a hard
cheese
whichis highly appreciated in Europe.Accordingto Rychlikand Bosset
3,4)
the most
potent
odorants of Gruyere cheese, whichare characterized by high
odor activity values, are sulfur-containing components derived from the amino
acidmetabolism of the ripening cultures, such as methanethiol, methional and
dimethyltrisulfide,
aswellas phenylaceticacidand 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. Theterpeneprofile 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),Buchinand co-workers
6),
and very
recently byCornuand co-workers (7).
Mariaca and co-workers
8)
investigated the volatile mono- and
sesquiterpenoids in different highland and lowland plant species. Theycould
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
andlowland
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) .
Toour knowledge this is the first study to investigate the influenceo frumen
fermentation on the degradation of monoterpenes
present
in a model plant
commonly
found on
pastures.
The results of the
invitro
studywillbe compared
to the
volatiles
found
in milk
and in cheese.
A
chiralstationaryphasewas employed to study whether the enantiomeric
ratiosofthe monoterpenes found
in
pasture
are maintained
in milk
and in cheese,
and to investigate the impact of the bovine rumen microflora on the
terpene
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Table
I. Aromacompoundsofa
lowland
Gruyerecheese
Compound Odor
descriptor
Cnc
activity
value
Methanethiol
Sulftirous
700
11300
Methional
Boiled
potato-like 99
495
Dimethyltrisulfide
Cabbage-like 136
54
Phenylacetic acid
Honey-like
7270 39
2-Methylbutanal
Malty 255
26
3-Methylbutanal
Malty
219 17
2-/3-Methylbutanoic acids Sweaty 81000 8.1
Acetic
acid
Vinegar-like,
pungent
298000 5.5
Butanoic acid Sweaty 97000
5.4
5-Decalactone
Swet,
coconut-like
1690
4.2
Propanoic acid
Fruity,
pungent
87000
2.9
Phenylacetaldehyde Honey-like 20 1
2,3-Diethyl-5-methylpyrazine Earthy 0.6 1
Methylpropanoic acid Sweaty, rancid 41000
0.5
)-2-Nonenal
Green 323
0.4
2-Ethyl-3,5-dimethylpyrazine Earthy
0.7
0.3
Hexanoic acid
Goat-like
28000 0.3
Data
are
from
references
3
and
4.
composition of cow parsnip
Heracleumsphondylium L. ) ,
which was selected as
model plant.
Experimental
Materials
The freshgrass
samples
originated from six alps in the Canton of
Vaud,
Switzerland,near the villages of LesMoulins and
L Et ivaz ,
at
altitudes
from
1400 to 1920 m. They
were
collected at the end of August 2003. Seven alps
located around LesMoulins(Canton of
Vaud,
Switzerland,
altitudes
of the alps:
1400 to 1900 m) provided the cow's milk (one sample
each),
and one Raclette-
type
cheeseinvestigated in thepresentstudy had
been
produced from thesame
milk. Cow parsnip
Heracleumsphondylium
L.) was collected twice, in mid-
August
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 milkand the cow parsnip
samples were
pooled to form composite samples.
The
samples were kept
frozen at -20 C
before
use.
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Rumen
fluidwas collectedfrom3 healthy cows aged between 5 and 8 years
withanartificialfistula connected to the rumen. The cows had been fedwithhay
ad
libitumfor two weeks prior to the study.
The composite
grass
sample (5 g) was roughly chopped and mixedwith
Mil l iQ
water (100 mL) in a Polytron PT 3000 mixer (Kinematica,
Littau,
Switzerland)
at 10000 rpm for 2 min. The suspension (2 mL) was dilutedwith
Mil l iQ
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 fluidaccording 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
Mil l iQ
water (100
mL )
and 2
mL
of thedilutionwere employed for
the P+T analysis.
Milk (20 mL) was directly pipetted into the sparger for
subsequent
P+T
analysis.The Raclette-type
cheese
wasfinelygrated and 5 g were weighed into a
beaker, mixedwith Mil l iQ 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
Dynamicheadspace
extraction (Purge + trap, P+T) using a
L SC
2000 P+T
system equippedwitha cryofocusing unit and a
trap
no. 8 (Tekmar,
Cincinnati,
OH )
was employed for the extraction of the monoterpenoids according to an
adapted method fromMariacaand 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
cooldown
-150
C;
desorb 4
min
at
240C;
inject 1.5 min
at 215
C;
bake
10 min
at 260
C;
transfer line to
G C
150 C.
Gas
chromatography coupled to
mass
spectrometry
( G C - M S )
for the
separation and analysis of the monoterpenes was conducted on an
Agilent
5890
Series II instrument (PaloA l to ,
C A )
equippedwitha 5971A
M SD
and achiral
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
(1.2mL/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 G C - M S
transfer line was set to 280 C, the M S D 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
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reference terpenes(Fluka, Buchs, Switzerland). The
terpene
metabolites were
tentatively identified based upon comparison of their mass spectra
with
commercial
mass spectrometrydatabasesand retention indices in the literature.
Single ion monitoring (SIM) of the ionsm/z93, 95, 119, 70, or 69 for the
monoterpenes and the
terpene
metabolites, respectively, was employed for
semiquantitation comparing their peak heights before and after incubationwith
rumen fluid. The ionsm/z136 and 93 were selected asqualifier 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-careneandP-myrcene and
the formation of menth-l-ene, 3,7-dimethyl-l,6-octadiene and 3,7-dimethyl-2-
octene. Except for the
correlation
the data were not
statistically
treated.
Resultsand
Discussion
The
grass
sample consisted of
plants
ofvariousfamiliesas described in (6),
i.e.
Apiaceae,
Asteraceae,Campanulaceae,Rosaceae,Fabaceae, Gerania
Lamiaceae, Plantaginaceae,
Ranunculaceae, Polygonaceae,
Rubiaceae,
also
Poaceae,thefamilyto
which
commongrassbelongs. 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 achiralstationary phase.
P
-Myrcene,
p-cymene, the two enantiomers (+)- and (-)-limonene, -)-P-ocimeneand (Z)-
P
-ocimene, y-terpinene and
-)-ot
-pinene were the predominant monoterpenes in
thegrasssamples analyzed (data not shown). Figure 1 depicts the structures of
the monoterpenes detected in the present study. Sabinene, cc-thujene,
(+)-a-
pinene, 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,andinthe 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 identifiedas 3,7-dimethyl-l,6-octadiene, 2,6-dimethyl-2,6-octadiene,
3,7-dimethyl-2-octene, and camphane. These monoterpenoids had beenabsentin
fresh grass. In the Raclette-typecheeseproducedfromthe milk basically the
same
monoterpenes were found as inmilk,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-dimethyl-
2-octene, camphane and menth-l-ene wereonlydetected in themilkandcheese
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In Flavor of Dairy Products; Cadwallader, K., et al.;ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
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TableII.
Monoterpenoids
detectedingrass,milk andRaclette-type
cheese
Compound
Grass
Milk
Raclette-type
cheese
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
2,6-Dimethyl-2,6-octadiene
/
Camphane
samples, however
not in
fresh grass.
The
presence
of
these
compounds
had
already been described in alpinemilkby
Buchin
and co-workers
6).
The enantiomeric ratios of selected monoterpenes were analyzed
to
examine
whether
the
ratios changedfrompastureto
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, inmilkand
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.
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Incubationof cow parsnip
with
rumen fluid
The
exclusive presence of the dimethyl octadienes, menth-l-ene and the
other tentatively identified compounds in
milk
andcheeseraised the hypothesis
that
thesecompounds might have been formed during rumen fermentation.Many
of
the monoterpenes shown in Figure show two orthreedouble 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 oronlyone double bond. Hydrogenation
duringrumen fermentation might beinvolved intheirformation.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,Heracleumsphondylium L., was selected as a
modelto investigate a potential correlation between the degradation of certain
monoterpenes and the formation of the above-mentioned compounds. Cow
parsnip belongs to thefamilyApiaceae,is found abundantly inpasturesandrich
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 ofincubation.
(Z)-(J-ocimene,
)-P
-ocimene, a-phellandrene and P-myrcene were
completelyor almost completely degraded,whilearound 40 % of (+)-limonene
and p-cymene, and 20 to 35 % of most other monoterpenes remained after 12 h
of
incubation.
During incubationwith rumen fluid the formation of the monoterpenoid
compounds
that
had been detected in
milk
and incheesewas observed. The
extent andrateof formation ofthesecompounds varied, as itcouldbe seen for
the degradation
of
the different monoterpenes.
Statisticallysignificant (p < 0.05) negative correlations were found for the
degradation of()-p-ocimeneand the co-eluting8-3-careneand the p-myrcene
and the formation of menth-l-ene, 3,7-dimethyl-l,6-octadiene and 3,7-dimethyl-
2-octene. )-P
-Ocimene,
8-3-carene andP-myrcene were almost completely
and rapidly degraded during the first 8 h ofincubation.Menth-l-ene increased
steadily
during the first 8 h ofincubation,then was
slowly
degradedfrom8 to
12 h of rumen fermentation.
After
12 h the menth-l-ene signal intensity dropped
rapidlyover the next 4 h as shownin Figure3.
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 incubationonly,
the onset of 3,7-dimethyl-2-octenecouldbe observed. The peak signal increased
rapidlyafter 12 hofrumen fermentation.
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700
600
S
' 500
(/)
>
a
400
S 2
100
0
Before incubation
After 12h incubation
1
1 il,
ll
Figure 2 Monoterpenoidsof
cow
parsnipbefore
andafter12
h ofincubation
with
rumenfluid
The formation of the newly formed compounds may be explained by
hydrogenation reactions by the rumenflora 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
-ocimeneyields 3,7-dimethyl-l,6-
octadiene, a shift
o f
the double bond at position
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-2,6-
octadiene
yields
3,7-dimethyl-2-octene.
Conclusions
The monoterpene composition offreshgrass,milk,and
cheese
was analyzed
by dynamic
headspace G C - M S .
It was shownthat milk and
cheese
revealed
additional
monoterpenes which had not been detected in the
grass
samples. A
model
plant, cow parsnip,
Heracleumsphondylium
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
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ZT)-B-Ocimene
8-3-carene
B-Myrcene
Menth-l-ene
3,7-Dimethyl-1,6-octadiene
3,7-Dimethyl-2-octene
2 4
8
12
16
24
Incubationtime h)
Figure
3.
Degradation
and
newformationof
selected
monoterpenoids
du
incubation
of
Heracleum
sphondylium L.with rumenfluid.
Hydrogenation
by
rumen flora
JL 0
t-Myrcene
(E)-p-Ocimene
(Z)-p-Ocimene
3,7-Dimethyl- 2,6-Dimethyl-
1,6-octadiene 2,6-octadiene
^Further
hydrogenation
3,7-Dimethyl-
2-octene
Figure4.
Hypothetic
schematicformationofselectedmonoterpenoid
byhydrogenation
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In Flavor of Dairy Products; Cadwallader, K., et al.;ACS Symposium Series; American Chemical Society: Washington, DC, 2007.
8/10/2019 Changes in Terpene Compo
11/11
133
menth-l-ene wereformed. It is hypothesized
that
p-myrcene, E)-and (Z
)-P-
ocimene are hydrogenatedduring incubation with rumen fluid to
yield
these
components.Theresultsshow
that
monoterpenes
are partiallydegradedduring
rumen fermentation and new compounds are formed, which are alsotransferred
into
milk
and
cheese.
Acknowledgments
The
authors
thank Roland Gauch and Bernard
Jeangros
for their help
collectingthe samples, KurtZbinden for providing the rumen
fluid
and Frigga
Dohme for useful discussions.
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