Changes in Terpene Compo

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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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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


/

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-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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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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German, M .P . ;

D'Angelo, V.;Marino, A.; Cannatelli,

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Delgado, B.; Palop, A.;Fernndez, P.S.; Periamo,

P . M . Eur.FoodRes.

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Rychlik,

M . ;

Bosset, J. O.

Int.DairyJ.

2001, 11, 895-901.

4. Rychlik,

M . ;

Bosset,J.O.

Int.DairyJ.

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