Investigation on aroma active photooxidative - ETH E

Diss. ETH No. 14862
Investigation on aroma active photooxidative degradation
products originating from dimethyl pentyl furan fatty acids
in green tea and dried green herbs
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the degree of
DOCTOR OF TECHNICAL SCIENCES
presented by
Isabelle Anna Sigrist
dipl. Lm.-Ing. ETH
born 07 April 1971
citizen of Meggen LU
accepted on the recommendation of
Prof. Dr. R. Amadò, examiner
Prof. Dr. P. Schieberle, co-examiner
Dr. G.G.G. Manzardo, co-examiner
Zurich 2002
Für meine Eltern
In Liebe und Dankbarkeit
Neue Ideen sind nur durch ihre
Ungewohnheit schwer verständlich.
Franz Marc, dt. Maler
DANKSAGUNG
An der erfolgreichen Durchführung der Dissertation sind immer mehrere Personen
beteiligt. Ich danke ganz herzlich...
... Prof. Dr. Renato Amadò für die Überlassung des Themas, die Unterstützung und die
Übernahme des Referates
... Dr. Giuseppe G.G. Manzardo für die fachliche Betreuung, die Unterstützung und die
Übernahme des Co-Referates
... Prof. Dr. Peter Schieberle für die Übernahme des Co-Referates
... Dr. Reto Battaglia von SQTS - Swiss Quality Testing Services (MGB) - für die
finanzielle Unterstützung
... Peter Oppliger AG und J. Carl Fridlin Gewürze AG für die grosszügige Bereitstellung
von Probenmaterial
... dem Deutschen Akademischen Austauschdienst (DAAD), Prof. Dr. H.M. Liebich,
Dr. Dr. H.G. Wahl, Josef Wöll und den Mitarbeitern des Zentrallabors der
Medizinischen Universitätsklinik Tübingen für die Unterstützung während meines
Forschungsaufenthaltes an der Eberhard-Karls Universität in Tübingen
... Dr. Ivo Niederer vom Kriminaltechnischen Dienst der Kantonspolizei St. Gallen für
die Benutzung des Ion Trap GC-MS
... Sandra Kürsteiner-Laube und Brigitte Jegge für die Synthese von Referenzsubstanzen
... Berit Abt für die Englisch-Korrekturen
... der ganzen Gruppe Lebensmittelchemie und -technologie für kleinere und grössere
Hilfestellungen
... den Semesterandinnen und Semesteranden Ueli von Ah, Valentine Cleusix, Sibilla
Delorenzi, Marianna Gulfi, Daniel Hiestand, Simon Kollaart und Marcel Leemann
für ihre Arbeiten
... meiner Familie für ihre grosse Unterstützung, ihr Verständnis und ihre Geduld
... Benedikt Koch für sein Verständnis und seine Energie
Part of this work has been published:
• Sigrist, I.A., Wunderli, B., Pompizzi, R., Manzardo, G.G.G., Amadò, R. (2000). Influence of
dimethyl furan fatty acid photooxidative degradation products on the flavour of green tea. In:
Schieberle, P., Engel, K.H. (eds.). Frontiers of Flavour Science. Deutsche Forschungsanstalt für
Lebensmittelchemie, Garching, D, 554-556.
• Sigrist, I.A., Manzardo, G.G.G., Amadò, R. (2000). Furan fatty acid photooxidative degradation
products in dried herbs and vegetables. Czech J. Food Sci. 18, 17-19; Sigrist, I.A., Manzardo,
G.G.G., Amadò, R. (2002). Furan fatty acid photooxidative degradation products in dried herbs and
vegetables. Chimia 56, 263-265.
• Sigrist, I.A., Manzardo, G.G.G., Amadò, R. (2001). Analysis of furan fatty acids in dried green
herbs using ion trap GC-MS/MS. In: Pfannhauser, W., Fenwick, G.R., Khokhar, S. (eds.).
Biologically-Active Phytochemicals in Food. The Royal Society of Chemistry, Cambridge, UK, 237240.
• Sigrist, I.A., Manzardo, G.G.G., Amadò, R. (2002). Aroma compounds formed from 3-methyl2,4-nonanedione under photooxidative conditions. Submitted to J. Agric. Food Chem.
I
TABLE OF CONTENTS
I
ABBREVIATIONS
II
SUMMARY
III ZUSAMMENFASSUNG
V
VII
IX
1
INTRODUCTION
1
2
LITERATURE REVIEW
3
2.1
Furan fatty acids
3
2.1.1
Structure and nomenclature
3
2.1.2
Occurrence
5
2.1.3
Relevance
8
2.2
Aroma active photooxidative degradation products of dimethyl pentyl
furan fatty acids
9
2.2.1
3-Methyl-2,4-nonanedione
9
2.2.2
2,3-Octanedione
14
2.2.3
Bovolide and Dihydrobovolide
18
2.2.4
Pentanal
24
2.2.5
2,3-Butanedione
25
II
Table of Contents
3
EXPERIMENTAL PART
27
3.1
Material
27
3.1.1
Sample material
27
3.1.2
Reference aroma compounds
27
3.1.3
Model mixture of reference aroma compounds
27
3.1.4
Dimethyl furan fatty acid standards
28
3.1.5
Chemicals
28
3.2
3.3
Synthesis of reference aroma compounds
29
3.2.1
3-Methyl-2,4-nonanedione
29
3.2.2
2,3-Octanedione
30
Analytical methods
30
3.3.1
Capillary gas chromatography (GC-FID)
30
3.3.2
Capillary gas chromatography-mass spectrometry
(GC-MS)
3.3.3
3.4
3.5
31
Capillary gas chromatography-ion trap mass spectrometry
(GC-MS/MS)
33
3.3.4
Capillary gas chromatography-infrared spectrometry (GC-IR)
34
3.3.5
Capillary gas chromatography-olfactometry (GC-O)
35
Extraction methods
36
3.4.1
Simultaneous distillation solvent extraction (SDE)
36
3.4.2
Accelerated solvent extraction (ASE)
37
Light exposure experiments
38
3.5.1
Sample preparation
38
3.5.2
Light exposure model system I
38
3.5.3
Light exposure model system II
39
Table of Contents
III
3.6
Oxidation experiments
40
3.6.1
Conditions in hexane
41
3.6.2
Conditions in methanol
41
4
RESULTS AND DISCUSSION
43
4.1
Analysis of furan fatty acids
43
4.1.1
Development of a method using ion trap gas chromatography-mass
spectrometry
4.1.2
Furan fatty acid content of green tea and dried green herbs and
vegetables
4.2
4.4
53
Oxidative stability of furan fatty acid photooxidative degradation
products
57
4.3.1
3-Methyl-2,4-nonanedione
57
4.3.2
2,3-Octanedione
67
Formation of furan fatty acid photooxidative degradation products in
green tea
4.5
50
Isolation of furan fatty acid photooxidative degradation products by
using micro simultaneous distillation solvent extraction
4.3
43
73
Formation of furan fatty acid photooxidative degradation products in
dried green herbs
80
5
CONCLUSION AND OUTLOOK
83
6
REFERENCES
87
IV
V
I
ABBREVIATIONS
A
area
AEDA
aroma extract dilution analysis
ASE
accelerated solvent extraction
BHT
2,6-di-tert-butyl-p-hydroxy-toluene
CHARM
combined hedonic and response measurement
DiMeF
dimethyl furan fatty acid(s)
eV
electron volt
FD
flavour dilution
FFA
furan fatty acid(s)
FID
flame ionisation detector
GC-O
gas chromatography-olfactometry
HMND
3-hydroxy-3-methyl-2,4-nonanedione
ID
inner diameter
ISTD
internal standard
MeF
monomethyl furan fatty acid(s)
MFD
meat flavour deterioration
MND
3-methyl-2,4-nonanedione
OAV
odour activity value
OC
on column
VI
Abbreviations
PE
polyethylene
r
correlation coefficient
RFID
peak area ratio, based on FID data
RI
retention index
RMS
peak area ratio, based on MS data
RMS/MS
peak area ratio, based on characteristic product ion
SDE
simultaneous distillation solvent extraction
SIDA
stable isotope dilution assay
TIC
total ion current
WOF
warmed over flavour
VII
II
SUMMARY
In the present work furan fatty acids (FFA) and their aroma active photooxidative
degradation products were investigated in dried green plant material used as foodstuff.
Green tea, tarragon, basil, savory, chervil, dill, chive, onion and leek were taken as
examples.
A new method using ion trap GC-MS/MS was developed for the analysis of FFA in food.
The application of the GC-MS/MS technique allowed a direct, fast and sensitive analysis
of FFA in the methyl ester extract of plant lipids. 12,15-Epoxy-13,14-dimethyl-eicosa12,14-dienoic acid (DiMeF(11,5)) and 10,13-epoxy-11,12-dimethyl-octadeca-10,12dienoic acid (DiMeF(9,5)) were the most abundant FFA found in the investigated
samples. The FFA contents were in the range of a few µg/g dry matter to more than
100 µg/g. To our knowledge, the occurrence of different classes of FFA in tarragon,
basil, savory, chervil, dill, onion and leek is reported for the first time in the present
work.
For the isolation of the volatile compounds from the samples a micro-SDE method was
adapted and critically assessed. The isolation procedure by micro-SDE was shown to be
suitable with respect to artefact formation, recovery and reproducibility. The
reproducibility of the method was assessed by using three internal standards. Concise
results could be obtained without performing replicate analyses.
The stability of the two diones 3-methyl-2,4-nonanedione (MND) and 2,3-octanedione
under photooxidative conditions was investigated in model experiments. Among the
light induced oxidation products of MND the five aroma compounds 2,3-butanedione,
2,3-octanedione, acetic acid, hexanoic acid and 3-hydroxy-3-methyl-2,4-nonanedione
(HMND) were identified. HMND was the main oxidation product. The odour can be
described as rubbery, earthy and plastic-like. This aroma compound was identified for
the first time in this work. Pentanal, acetic acid and hexanoic acid were the aroma active
VIII
Summary
oxidation products derived from 2,3-octanedione. Based on the results of the model
experiments, other pathways for the formation of FFA photooxidative degradation
products than the ones described in the literature have to be taken into account.
Light exposure experiments with green tea, dried herbs and vegetables were carried out
to investigate the photooxidative degradation products of FFA in complex food systems.
In green tea the formation rate of the aroma compounds during a long period of light
exposure (20 or 25 days) in air or in oxygen atmosphere was investigated. 2,3Butanedione showed only slight changes. The formation of MND and HMND showed a
maximum after two days of light exposure, followed by a constant slight decrease.
Pentanal, 2,3-octanedione, 2,3-dimethylnona-2,4-dien-4-olide (bovolide) and 2,3dimethylnon-2-en-4-olide (dihydrobovolide) increased continuously during the time of
light exposure. The major increase in the amounts of these aroma compounds occurred
during the first two days. The formation of the seven aroma compounds was in
accordance with the decrease of the two main pentyl DiMeF in the green tea during light
exposure. After two days of light exposure, approximately 50 % of the pentyl DiMeF
had reacted; after 20 days approximately 10 % of the initial amount were still present.
The investigated herbs (tarragon, basil, savory, chervil, dill and chive) as well as leek
and onion showed to be differently susceptible to light exposure. The amounts of
pentanal, 2,3-butanedione, 2,3-octanedione, bovolide and dihydrobovolide increased
during the time of light exposure. MND and HMND were only detected in tarragon,
chervil, dill, chive and leek after light exposure.
IX
III ZUSAMMENFASSUNG
In der vorliegenden Arbeit wurden Furanfettsäuren (FFA) und deren aromaaktive,
photooxidativ gebildete Abbauprodukte in grünem getrockneten Pflanzenmaterial, das
als Lebensmittel verwendet wird, untersucht. Als Beispiele wurden Grüntee, Estragon,
Basilikum, Bohnenkraut, Kerbel, Dill, Schnittlauch, Zwiebel und Lauch gewählt.
Für die Analyse der FFA in den gewählten Proben wurde eine Methode mit Ion Trap
GC-MS/MS entwickelt. Die Anwendung der GC-MS/MS Technik erlaubte eine direkte,
schnelle und empfindliche Analyse der FFA im Methylesterextrakt der Pflanzenlipide.
Die zwei am häufigsten in den Proben vorkommenden FFA waren die 12,15-Epoxy13,14-dimethyleicosa-12,14-diensäure (DiMeF(11,5)) und die 10,13-Epoxy-11,12dimethyloctadeca-10,12-diensäure (DiMeF(9,5)). Die Furanfettsäurengehalte lagen
zwischen einigen µg/g Trockensubstanz bis über 100 µg/g. Nach unserem Wissen wird
hier zum ersten Mal über ein Vorkommen von verschiedenen Furanfettsäureklassen in
Estragon, Basilikum, Bohnenkraut, Dill, Zwiebel und Lauch berichtet.
Für die Isolierung der flüchtigen Stoffe aus den Proben wurde eine Mikro-SDE
angepasst und kritisch beurteilt. Die Isolierung mittels Mikro-SDE erwies sich bezüglich
Artefaktbildung, Wiederfindung und Reproduzierbarkeit als geeignet. Die Reproduzierbarkeit der Methode wurde durch Anwendung von drei internen Standards überprüft.
Damit konnten ohne Mehrfachanalysen präzise Resultate erhalten werden.
Die Stabilität der beiden Dione 3-Methyl-2,4-nonandion (MND) und 2,3-Octandion
unter photooxidativen Bedingungen wurde in Modellexperimenten untersucht. Unter
den lichtinduzierten Oxidationsprodukten von MND wurden die fünf Aromastoffe 2,3Butandion, 2,3-Octandion, Essigsäure, Hexansäure und 3-Hydroxy-3-methyl-2,4nonandion (HMND) identifiziert. HMND war das Hauptoxidationsprodukt. Der Geruch
von HMND lässt sich als gummiartig, erdig und plastikartig beschreiben. Dieser
Aromastoff wurde in dieser Arbeit zum ersten Mal identifiziert. Pentanal, Essigsäure
X
Zusammenfassung
und Hexansäure gehörten zu den aromaaktiven Oxidationsprodukten von 2,3Octandion. Basierend auf den Resultaten aus diesen Modellexperimenten müssen
andere Bildungswege für die photooxidativen Abbauprodukte der Furanfettsäuren in
Betracht gezogen werden als in der Literatur beschrieben.
Mit Hilfe von Belichtungsexperimenten mit Grüntee, getrockneten Kräutern und
getrocknetem Gemüse wurde die Bildung der photooxidativen Abbauprodukte der FFA
im komplexen System Lebensmittel untersucht. In Grüntee wurde die Bildungsrate der
Aromastoffe während einer längeren Lichtexposition (20 oder 25 Tage) an der Luft oder
in Sauerstoffatmosphäre ermittelt. 2,3-Butandion zeigte lediglich geringe Veränderungen. MND und HMND zeigten ein Maximun nach zweitägiger Belichtungsdauer und
danach eine konstante leichte Abnahme. Pentanal, 2,3-Octandion, 2,3-Dimethylnona2,4-dien-4-olid (Bovolid) und 2,3-Dimethylnon-2-en-4-olid (Dihydrobovolid) nahmen
während der ganzen Belichtungsdauer zu, wobei die Hauptmengen während den ersten
zwei Tagen gebildet wurden. Die Bildung der sieben Aromastoffe konnte in Beziehung
mit der Abnahme der beiden Hauptfuranfettsäuren in Grüntee während der Belichtung
gebracht werden. Nach zwei Tagen Belichtung hatten ca. 50 % der Dimethylpentylfuranfettsäuren reagiert; ungefähr 10 % der Anfangsmenge waren nach 20 Tagen
Belichtung immer noch vorhanden.
Die untersuchten Kräuter (Estragon, Basilikum, Bohnenkraut, Kerbel, Dill und
Schnittlauch) sowie Lauch und Zwiebeln waren unterschiedlich lichtanfällig. Die
Menge an Pentanal, 2,3-Butandion, 2,3-Octandion, Bovolid und Dihydrobovolid nahm
generell während der Belichtungszeit zu. MND und HMND konnten nach der
Belichtung nur in Estragon, Kerbel, Dill, Schnittlauch und Lauch nachgewiesen werden.
1
1
INTRODUCTION
Furan fatty acids (FFA) are minor components of the lipid fraction and occur widely in
different plants, vegetable oils, seafood and mammals. It is assumed that FFA are
common constituents of plants and accumulate in animal tissue through the food chain
(Hannemann et al., 1989). Recently, this special class of lipid compounds gained in
importance with the identification of FFA as precursors of light induced aroma
compounds (Guth and Grosch, 1991; Pompizzi et al., 2000). Some of the aroma active
FFA photooxidative degradation products are supposed to contribute significantly to the
flavour of soya-bean oil (Guth and Grosch, 1989), butter and butter oil (Grosch et al.,
1992), green tea (Guth and Grosch, 1993a), dry parsley (Masanetz and Grosch, 1998),
dry spinach (Masanetz et al., 1998) and anchovy (Triqui and Reineccius, 1995a, 1995b).
The present investigation intended to contribute to an extended insight of FFA and their
aroma active photooxidative degradation products in dried green plant material used as
foodstuff. The studies were undertaken with green tea, dried green herbs and some
selected vegetables. The investigation focused on:
• Analysis of FFA
• Light exposure experiments to induce the formation of FFA photooxidative
degradation products
• Analysis of the aroma active FFA photooxidative degradation products in the
selected samples
Established procedures to analyse FFA in biological samples are all time-consuming or
require uncommon equipment. Therefore a method had to be developed for an easier
practicable and faster analysis of FFA in the selected samples.
2
Introduction
In order to study the aroma active photooxidative degradation products of FFA it is
necessary to isolate the volatile substances from the nonvolatile plant material. The
sample preparation is the most critical step in the entire analytical process of the
investigation of volatile compounds (Schreier, 1984). Various isolation techniques have
been developed (see e.g. in Schreier, 1984 or Maarse and Grosch, 1996). In this part of
the investigation it was therefore very important to choose and assess a suitable method
for the isolation of aroma compounds and to carefully adapt it to the corresponding
problem.
Light exposure experiments in model systems were conducted in order to complement
and to extend the understanding of the formation and possible pathways of products
derived from FFA under photooxidative conditions. Finally light exposure experiments
with green tea and dried herbs and vegetables should result in a better comprehension of
light induced processes in the complex food system.
3
2
LITERATURE REVIEW
2.1
Furan fatty acids
2.1.1
Structure and nomenclature
Furan fatty acids (FFA) (Fig. 1) are characterised by the presence of a di-, tri- or
tetrasubstituted furan ring in the molecule. They differ in the degree of methyl
substitution at position 3 and 4 and in the length of the alkyl carboxyl chain and the alkyl
chain. The most common members of the dimethyl substituted FFA (DiMeF) are the
propyl FFA (bearing a terminal propyl group) and the pentyl FFA (bearing a terminal
pentyl group).
R2
R1
4
3
5
H3C(H2C)n
2
O
(CH2)mCOOH
R1 & R2 = H
R1 & R2 = CH3
R1 = CH3 & R2 = H
n = 2: propyl FFA
n = 4: pentyl FFA
Figure 1: Structure of furan fatty acids
For the nomenclature two different abbreviation systems are used. Glass et al. (1975)
named the FFA F1, F2,... based on the sequence of elution in the GC. Rahn et al. (1981)
suggested a self-explanatory abbreviation nomenclature. The FFA are termed as
F(m+1, n+1), MeF(m+1, n+1) or DiMeF(m+1, n+1). F stands for the furan ring, the
prefix Me or DiMe for a mono- or dimethyl substitution at position 3 or at position 3 and
4, respectively. The affix (m+1, n+1) indicates the chain lengths of the alkyl carboxyl
group at position 2 and the alkyl group at position 5. In this thesis, the abbreviated form
4
Literature Review
by Rahn et al. (1981) will be used. Tab. 1 gives an overview of the nomenclature and the
abbreviations of the most common FFA.
Table 1: Nomenclature of furan fatty acids
systematic nomenclature
10,13-epoxy-11,12-dimethyl-
Glass et al.
Rahn et al.
(1975)
(1981)
R1
R2
m
n
F1
diMeF(9,3)
CH3
CH3
8
2
F2
MeF(9,5)
CH3
H
8
4
F3
diMeF(9,5)
CH3
CH3
8
4
F4
diMeF(11,3)
CH3
CH3
10
2
F5
MeF(11,5)
CH3
H
10
4
F6
diMeF(11,5)
CH3
CH3
10
4
F7
MeF(13,5)
CH3
H
12
4
F8
DiMeF(13,5) CH3
CH3
12
4
hexadeca-10,12-dienoic acid
10,13-epoxy-11-methyloctadeca-10,12-dienoic acid
10,13-epoxy-11,12-dimethyloctadeca-10,12-dienoic acid
12,15-epoxy-13,14-dimethyloctadeca-12,14-dienoic acid
12,15-epoxy-13-methyleicosa-12,14-dienoic acid
12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid
14,17-epoxy-15-methyldocosa-14,16-dienoic acid
14,17-epoxy-15,16-dimethyldocosa-14,16-dienoic acid
In nature, DiMeF are predominant, whereas monomethyl substituted FFA (MeF) are
generally present in significantly lower concentrations. Natural occurrence of non
methyl substituted FFA is uncertain. Yurawecz et al. (1995) determined the non methyl
substituted FFA as oxidation products of conjugated linoleic acids in model
experiments. This class of FFA will not be discussed in detail in this work.
Literature Review
2.1.2
5
Occurrence
The analysis of different lipid fractions have revealed FFA to be constituents of
cholesteryl esters, triglycerides and phospholipids, depending on the samples
investigated. In freshwater fish for example, the FFA occurred in triglycerides and
esterified to cholesterol (Glass et al., 1974), whereas in human blood plasma and bovine
blood plasma (Puchta et al., 1988) and in sugar cane (Saccharum spec.) cells
(Scheinkönig and Spiteller, 1993) they mainly occurred in phospholipids.
F(8,6) was the first fatty acid of the furanoid type to be reported. Morris et al. (1966)
claimed that F(8,6) occurs in the Exocarpus seed oil. This result could not be confirmed
by Gunstone et al. (1978) who supposed this hexyl FFA to be an artefact from an
associated oxygenated acetylenic fatty acid. Investigations by Glass et al. (1974, 1975,
1977) showed a wide occurrence of different FFA in freshwater fish. Lower percentages
than in freshwater fish have been found also in various species of marine fish by
Gunstone et al. (1976, 1978), Gunstone and Wijesundera (1978), Scrimgeour (1977),
Yoshioka (1981), Ota and Takagi (1989a, 1989b, 1990, 1991, 1992) and Itabashi et al.
(1994, 1995). Wahl et al. (1994) and Wahl (1998) investigated the occurrence of FFA in
fish oil and fish oil compounds. Low levels of FFA have been reported to occur in other
aquatic species as well, such as in the tissues of crayfish (Okajima et al., 1984; Ishii et
al., 1988a, 1989a; Dembitsky and Rezanka, 1996), octopus (Gunstone et al., 1978), sea
squirt (Yoshioka, 1981), scallops (Yoshioka, 1981; Dembitsky and Rezanka, 1996), soft
corals (Groweiss and Kashman, 1978) and sponges (Ciminiello et al., 1991; Prinsep et
al., 1994). The list can be expanded to amphibia such as bullfrog, and reptiles such as
turtles as examples (Ishii et al., 1988a).
The occurrence of FFA in plants seems to be of greater importance. In the latex of the
rubber tree Hevea brasiliensis MeF(9,5) has been identified as the main component of
the lipid fraction (Hasma and Subramaniam, 1978; Lie Ken Jie and Sinha, 1980).
Different FFA were detected as minor components in numerous other plants
(Hannemann et al., 1989), in yeast (Hannemann et al., 1989) and in algae (Kazlauskas
et al., 1982; Hannemann et al., 1989; Batna et al., 1993; Itabashi et al., 1995). The levels
were higher in photosynthetic tissues than in the other parts of the plants. Recently, the
occurrence of FFA in green tea (Guth and Grosch, 1993a), dried spinach (Masanetz et
al., 1998) and dried parsley (Masanetz and Grosch, 1998) was reported.
6
Literature Review
FFA have been identified as minor components in blood, liver, muscle and lipid tissue
and in milk of rats (Gorst-Allman et al., 1988), cattle (Jandke and Spiteller, 1988;
Schödel and Spiteller, 1987; Puchta et al., 1988; Guth and Grosch, 1992) and humans
(Puchta et al., 1988; Puchta and Spiteller, 1988; Wahl et al., 1995). This indicates an
ubiquitous distribution of FFA in nature. However, it remains an open question if FFA
are synthesised de novo or introduced by the diet in mammals.
As for the origin of FFA in fish, recent findings indicate that they possibly arise not only
from marine plants but also from intestinal bacteria of fishes. Shirasaka et al. (1995,
1997) investigated MeF in marine bacteria such as Shewanella putrefaciens and
Pseudomonas fluorescens. Very recently, MeF was also identified in a Bacillus sp.
(Carballeira et al. 2000). In Tab. 2 all FFA detected up to now are listed in groups.
Table 2: Occurrence of furan fatty acids
FFA
first detected in
reference
MeF(9,3)
brown alga
Kazlauskas et al. (1982)
MeF(11,3)
salmon roe
Ishii et al. (1988b)
DiMeF(7,3)
crayfish
Ishii et al. (1988a)
DiMeF(9,3)
northern pike
Glass et al. (1974)
DiMeF(11,3)
northern pike
Glass et al. (1974)
DiMeF(13,3)
different marine and
Gunstone et al. (1978)
propyl
freshwater fish
DiMeF(15,3)
fish oil
Wahl et al. (1994)
MeF(9,4)
crayfish
Ishii et al. (1988a)
DiMeF(9,4)
crayfish
Ishii et al. (1988a)
DiMeF(11,4)
salmon roe
Ishii et al. (1988b)
butyl
Literature Review
7
Table 2: Occurrence of furan fatty acids (cont.)
FFA
first detected in
reference
MeF(3,5)
crayfish
Ishii et al. (1988a)
MeF(5,5)
butter and butter oil
Guth and Grosch (1992)
MeF(7,5)
crayfish
Ishii et al. (1988a)
MeF(9,5)
northern pike
Glass et al. (1974)
MeF(11,5)
northern pike
Glass et al. (1974)
MeF(13,5)
northern pike
Glass et al. (1974)
MeF(15,5)
crayfish
Ishii et al. (1988a)
DiMeF(3,5)
soft corals
Groweiss and Kashman (1978)
DiMeF(5,5)
crayfish
Ishii et al. (1988a)
DiMeF(7,5)
crayfish
Okajima et al. (1984)
DiMeF(8,5)
crayfish
Ishii et al. (1988a)
DiMeF(9,5)
northern pike
Glass et al. (1974)
DiMeF(10,5)
crayfish
Ishii et al. (1988a)
DiMeF(11,5)
northern pike
Glass et al. (1974)
DiMeF(12,5)
crayfish
Ishii et al. (1988a)
DiMeF(13,5)
northern pike
Glass et al. (1974)
DiMeF(15,5)
mollusc
Dembitsky and Rezanka (1996)
F(8,6)
Exocarpus seed oil
Morris et al. (1966)
MeF(9,6)
crayfish
Ishii et al. (1988a)
pentyl
hexyl
8
Literature Review
Table 2: Occurrence of furan fatty acids (cont.)
FFA
first detected in
reference
DiMeF(7,6)
crayfish
Ishii et al. (1988a)
DiMeF(9,6)
crayfish
Ishii et al. (1988a)
DiMeF(11,6)
crayfish
Ishii et al. (1988a)
MeF(5,7)
crayfish
Ishii et al. (1988a)
MeF(11,7)
crayfish
Ishii et al. (1988a)
DiMeF(7,7)
crayfish
Ishii et al. (1988a)
MeF(9,1)
brown alga
Kazlauskas et al. (1982)
F(2,14)∆8,11,14,17
marine sponge
Ciminiello et al. (1991)
F(2,20)∆9,19
marine sponge
Ciminiello et al. (1991)
heptyl
miscellaneous
2.1.3
Relevance
The role of the FFA in biological systems remains more or less obscure. According to
Batna and Spiteller (1994a, 1994b) FFA could be important in plant defence through the
products resulting from their oxidative degradation. Further it is assumed that FFA may
serve as antioxidants because of their hydroxyl radical scavenging activity (Ishii et al.,
1989b; Okada et al., 1990a, 1996) and their inhibitory activity on horseradish peroxidase
(Fuchs and Spiteller, 1999). Okada et al. (1990b) examined an inhibitory effect on the
haemolysis of erythrocytes induced by singlet oxygen and found the FFA to be effective
quenchers. Additionally, FFA have been reported to have inhibitory effects on blood
platelet aggregation (Graff et al., 1984) and on bacterial urease activity (Rosenblat et al.,
1993) and to have potential antitumor activity (Isoda et al., 1993). One non methyl
substituted FFA (F(5,6)) also showed a toxic effect on Tripanosoma brucei, which
causes the sleeping sickness (Doering et al., 1994).
Literature Review
9
FFA are considered as precursors of some potent aroma compounds. 3-Methyl-2,4nonanedione (MND) was the first aroma compound that was recognised to be a
photooxidative degradation product of pentyl DiMeF (Guth and Grosch, 1991).
Moreover, Sarelse et al. (1994) put forth the hypothesis that 2,3-dimethylnona-2,4-dien4-olide (bovolide) and 2,3-dimethylnon-2-en-4-olide (dihydrobovolide) were formed by
photooxidation of FFA. This could be confirmed experimentally by Pompizzi et al.
(1997). The importance of pentyl DiMeF as precursors of flavour compounds was
systematically investigated by Pompizzi (1999) in his doctoral thesis. Photooxidative
and autoxidative model experiments were carried out with DiMeF(9,5) methyl ester.
Among the oxidation products, eight aroma compounds could be identified, namely
pentanal, 2,3-butanedione, 2,3-octanedione, MND, bovolide, dihydrobovolide,
hexanoic acid and valeric acid. Model experiments with DiMeF(11,3) showed the
formation of the analogous aroma compounds through oxidation: propanal, 2,3hexanedione, 3-methyl-2,4-heptanedione, 2,3-dimethylhepta-2,4-dien-4-olide and 2,3dimethylhept-2-en-4-olide. In the following chapters an overview of the six aroma
compounds which are relevant for the present work is given.
2.2
Aroma active photooxidative degradation products
of dimethyl pentyl furan fatty acids
2.2.1
3-Methyl-2,4-nonanedione
MND is a β-diketone, of which the keto and enol tautomers can easily be separated by
GC using a nonpolar capillary column (Masur et al., 1987). Guth and Grosch (1989)
observed a major and a minor peak in the gas chromatogram obtained using a SE-30
capillary as stationary phase. The increase in the base-line between the two peaks was
caused most likely by changes in the keto-enol equilibrium during the GC analysis.
The first odour descriptor for MND was lard-like, strawy and fruity (Guth and Grosch,
1989). Additional odour qualities perceived at the sniffing port were sweet (Triqui and
Reineccius, 1995a), hay-like (Masanetz et al., 1998) and green (Kumazawa and Masuda,
1999). Often only one of these descriptors or a combination are used in the literature. A
differing odour descriptor for MND is anis-like and brackish (Schlüter et al., 1996). The
odour thresholds in different media are given in Tab. 3.
10
Literature Review
Table 3: Odour thresholds for MND in different media
medium
threshold
reference
air
0.007-0.014 ng/l
Guth and Grosch (1989)
0.06 ng/l (keto form)
Pompizzi (1999)
0.05 ng/l (enol form)
Pompizzi (1999)
0.03 µg/kg (orthonasal)
Masanetz and Grosch (1998)
0.02 µg/kg (retronasal)
Guth and Grosch (1993a)
15-30 µg/kg (orthonasal)
Guth and Grosch (1989)
1.5 µg/kg (retronasal)
Guth (1991)
1.5 µg/kg
Guth and Grosch (1993a)
water
oil
cellulose
Based on an aroma extract dilution analysis (AEDA), Guth and Grosch (1989, 1990a)
identified MND as the most important odorant of soy bean oil, which had a „beanystrawy“ off-flavour after storage in daylight for 30 days. Quantification by stable isotope
dilution assay (SIDA) confirmed the importance of MND in the so called reversion
flavour of soy bean oil. The calculated odour activity value (OAV, ratio of the
concentration of the aroma compound to its threshold) indicated that MND was the most
flavour-active compound after 48 h of storage in daylight (Guth and Grosch, 1990b).
Detection of FFA as precursors of MND in soy bean oil clarified the correlation between
MND, FFA and the light-induced deterioration of soy bean oil (Guth and Grosch, 1991).
However, findings by Kao et al. (1998) did not support the theory that FFA or MND
contribute strongly to the reversion flavour. Guth and Grosch (1999) confirmed their
suggestion in a letter to the editor. The potent odorants in standardised, enzymatically
hydrolysed and deoiled soybean lecithins were systematically characterised by Stephan
and Steinhart (1999a). MND was found to be an important odorant on the basis of the
flavour dilution (FD) factor and combined hedonic and response measurement
(CHARM) values. However, MND was also only one of a large number of odorants that
are responsible for the overall lecithin aroma. This suggestion was confirmed by
quantification and calculation of the nasal and retronasal OAVs (Stephan and Steinhart,
Literature Review
11
1999b). Though MND showed a potent retronasal OAV, the compound had no
outstanding sensory characteristics in soybean lecithin. The authors therefore concluded
that MND cannot be the character impact compound that is solely responsible for the
main strawy and grain-like odour of soybean lecithins.
Investigations by Grosch et al. (1992) showed that exposure of butter and butter oil to
fluorescent light changed the flavour from buttery, sweet and acidulous to green, strawy
and fatty. A comparative AEDA indicated that this change was mainly due to the
production of MND. This result has been completed with the identification of FFA in
butter and butter oil as precursor of MND causing the off-flavour (Guth and Grosch,
1992).
AEDA and calculation of OAV revealed MND as one of the most significant odorants
of green tea powder and brew (Guth and Grosch, 1993a). This result was associated to
the occurrence of FFA in green tea. Already some years ago, Horita and Hara (1986)
reported on an unknown light-produced aroma compound of green tea. The mass
spectral characteristics of this compound were in agreement with those of MND. MND
was detected in black tea as well, and evaluated by AEDA and calculation of OAV (Guth
and Grosch, 1993b). Although the dione occurred in similarly high concentration as in
green tea, it was not considered to be a key aroma compound since other potent aroma
compounds masked the hay-like odour. Recently Kumazawa and Masuda (1999)
identified potent odorants in Japanese green tea (Sencha) based on AEDA. MND was
not found to belong to the eight most potent odour compounds. The authors mentioned
that the Japanese green tea is manufactured according to a process different from the one
used for Chinese green tea. Guth and Grosch (1993a) investigated Chinese green tea
powder and assumed that the MND was formed during the processing of green tea.
Masanetz and Grosch (1998) showed by AEDA and calculation of OAV that MND was
mainly responsible for the hay-like off-flavour of dried parsley, which developed during
drying and storage. In these samples FFA were detected as precursors as well. Dry
spinach was investigated by Masanetz et al. (1998). The detection of FFA and MND led
to the conclusion that the hay-like off-flavour was caused by an oxidative degradation of
FFA to MND. However, in extracts of cooked spinach leaves, MND was only detected
in traces (Näf and Velluz, 2000). Depending on the concentration, a contribution to the
pleasant and warm hay-cereal-flour-like and buttery, green-woody odour was not
excluded. An interesting fact was the detection of (E)-3-methyl-2-nonen-4-one in the
12
Literature Review
cooked spinach leaves as a heavy, tenacious compound exhibiting a green, stalk, lovagelike note. Its structure is related to MND, and it could be a degradation product of
bovolide (see chapter 2.2.3).
So far, MND has not only been detected in plants, but also in fish. Triqui and Reineccius
(1995a, 1995b) investigated the flavour development in anchovy during ripening by
means of AEDA. MND was reported to be an important flavour component. Since it was
identified in anchovy just after salting, a formation following evisceration was assumed.
Natural sensitisers in fish may generate singlet oxygen which may then react with FFA
present in anchovy. Schlüter et al. (1996) used GC-O, AEDA and CHARM to analyse
aroma extracts of steamed fillets of carp fed either with zooplankton or mainly with
wheat. MND was perceived only in the sample fed with plankton. Based on the FD factor
and the CHARM value, MND was not considered to be important. Somewhat later,
MND was mentioned as a potent odorant in boiled carp fillet based on FD factors
(Schlüter et al., 1999). Based on the FD factor as well, MND was reported to be a potent
odorant in boiled trout (Milo and Grosch, 1993).
Two different pathways for the photooxidative formation of MND have been described
in the literature. Guth and Grosch (1991) proposed an ene-reaction of a pentyl DiMeF
with singlet oxygen directly to a hydroperoxide. β-scission of the hydroperoxy-group
and subsequent further cleavages resulted in the keto-enol form of MND and a ketene
(Fig. 2). Pompizzi et al. (2000) suggested a cycloaddition of singlet oxygen to
DiMeF(9,5) to a bicyclic furan endoperoxide. Rearrangements then resulted in the βketo-enol ester that was transformed by hydrolysis to MND (Fig. 3).
Literature Review
13
R
O
+ 1O2
R
OH
O
O
R = (CH2)nCOOCH3
n = 7 or 9
O=C=CH
+
O
O
OH O
Figure 2: Proposed pathway for the photooxidative formation of MND
(Guth and Grosch, 1991)
R2
R1
+ 1O2
R2
R1
O
O
O
O
R1 = (CH2)3CH3
R2 = (CH2)7COOCH3
R1
R2
O
O
O
H 2O / H +
+ HO
R1
R1
O
O
O
OH
R2
O
Figure 3: Proposed pathway for the photooxidative formation of MND
(Pompizzi et al., 2000)
R
14
2.2.2
Literature Review
2,3-Octanedione
2,3-Octanedione is an aroma compound which odour has been described in many
different ways in the literature. Winter et al. (1976) used the descriptor caramel-like and
sweet. The odour has also been described as sweet and fruity (Suzuki, 1985), fragrant and
meaty (Ames and Elmore, 1992), fatty (Sutherland and Ames, 1995), green, ketoney,
grassy and strawy (Braggins, 1996), hay-like and fruity (Pompizzi et al., 2000), pungent
and sour (Hartvigsen et al., 2000) and mushroom-like (Ulrich et al., 2001). The odour
threshold in air is 0.09 ng/l (Pompizzi, 1999), in oil orthonasal 2.0 mg/kg (Pompizzi,
1999) and in water orthonasal 0.11 mg/kg (Sigrist et al., 2000).
2,3-Octanedione was detected as volatile compound in tobacco (Demole and Berthet,
1972), dried mushroom (Vidal et al., 1986), bell peppers fruits (Matsui et al., 1997),
apricot pureé (Bolzoni et al., 1990), prickly pear juice (Di Cesare and Nani, 1992) and
black elder flowers (Mazza, 2001). None of these studies contain any information of a
possible contribution of 2,3-octanedione to the aroma. In heated blackberry juices
(Georgilopoulos and Gallois, 1987) and in anise (Kollmannsberger et al., 2000) the
diketone was identified but recognised as a compound with no odour intensity. In potato
flesh, 2,3-octanedione was identified by Oruna-Concha et al. (2001) as a volatile but not
as a key aroma compound. 2,3-Octanedione was detected in traces in the volatile
constituents of used frying oil (Takeoka et al., 1996). Traces present in soybean oil and
corn oil underwent quantitative changes during storage due to oxidation (Wu and Chen,
1992). In dried green plant material 2,3-octanedione was detected in the headspace of
sun-cured fescue hay (Mayland et al., 1997) and in Oolong tea (Suxian et al., 1998).
Additionally, Horita and Hara (1986) reported on an unknown volatile compound of
green tea exposed to light, which mass spectral characteristics fitted to 2,3-octanedione.
In cereals, 2,3-octanedione has often been described as a product of lipid degradation
with no further explanation to its formation. Pfannhauser (1990) identified the dione in
triticale treated by cooking extrusion, and Hwang et al. (1994) in extruded wheat flour.
Bailey et al. (1994a) identified it in an extruded product of a mixture of whey protein
concentrate with corn meal, and Parker et al. (2000) in extruded oat flour. Nijssen et al.
(1996) found 2,3-octanedione in soy and unfermented soy products, and Aaslyng et al.
(1998) in the headspace of hydrolysed soy protein. 2,3-Octanedione was also present in
wheat bread crust (Chang et al., 1995) and in sour dough bread (Seitz et al., 1998). In
Literature Review
15
yeast extract 2,3-octanedione has been stated as an aroma compound (Ames and Elmore,
1992). Recently, 2,3-octanedione was detected by GC-O (CHARM) as a key compound
in boiled asparagus and a contribution to the aroma was assumed (Ulrich et al., 2001).
The occurrence of 2,3-octanedione in meat and meat products seems to be of much
greater significance than in plants. 2,3-Octanedione was first detected in beef (roast
beef) by Liebich et al. (1972), in mutton (cooked mutton) by Nixon et al. (1979), in
poultry (roasted chicken) by Noleau and Toulemonde (1986), in chevon (cooked
chevon) by Lamikanra and Dupuy (1990), in pork (uncured pork) by Ramarathnam et
al. (1991a) and in venison (deer) by Clarke et al. (1995), who in addition reported 2,3octanedione to be a suitable marker compound to predict the duration of frozen storage
of meat. Young et al. (1997) investigated the influence of the diet on volatiles of lamb
fat and found 2,3-octanedione to be an excellent indicator of a pasture diet. Already
earlier Suzuki and Bailey (1985) reported on very high contents of 2,3-octanedione in the
volatiles from ovine fat of animals finished on clover, compared to those of animals
finished on corn. Later, Bailey et al. (1994b) investigated the influence of finishing diets
on lamb flavour. Meat from animals finished on corn grain was milder than that of
animals finished on forage, and 2,3-octanedione was one of the volatile compounds
correlating with flavour strength. In beef, 2,3-octanedione was reported to be an
indicator for a pasture diet (Larick et al., 1987) as well. However, in a study with foragefed goats, 2,3-octanedione was less prominent in the cooked meat (Lamikanra and
Dupuy, 1990).
In the context of meat flavour quality, a possible correlation of 2,3-octanedione and the
so called warmed-over flavour or WOF (also referred to as meat flavour deterioration,
or MFD) has to be mentioned. WOF is the rapid development of an off-flavour in
refrigerated cooked meat described as rancid or stale (Tims and Watts, 1958). Although
the precise mechanisms of the formation of WOF have not yet been elucidated, the
generally accepted theory states that WOF is caused by the oxidation of membrane
phospholipids, catalysed by an ionic form of iron (Pearson and Gray, 1983; Love, 1987).
Among some other aroma compounds, 2,3-octanedione was first proposed by St. Angelo
et al. (1987a) as WOF marker due to its high correlation with WOF formation in beef.
Further studies on WOF were performed with 2,3-octanedione as marker in cooked
turkey rolls (Wu and Sheldon, 1988), beef (Vercellotti et al., 1988), cooked chevon
16
Literature Review
(Lamikanra and Dupuy, 1990), beef patties stored under vacuum (Spanier et al., 1992a,
1992b) and freeze-dried lean beef (Thongwong et al., 1999). However, many studies on
WOF exist in which 2,3-octanedione has not even been mentioned.
Some results of investigations on uncured and cured meat are noteworthy. Ramarathnam
et al. (1991a) found 2,3-octanedione to be present in appreciable levels in uncured pork
but absent in cured pork. The same result was obtained with uncured and cured beef and
chicken (Ramarathnam et al., 1991b). However, Dirinck et al. (1997) and Ruiz et al.
(1998, 2001) detected the dione in dry-cured Iberian ham and Timón et al. (2001) in the
subcutaneous fat from dry-cured hams. Ruiz et al. (1999) observed that the volatile
compounds of dry-cured ham were affected by the length of the curing process; 2,3octanedione decreased significantly during the ripening process.
With regard to meat products, 2,3-octanedione was detected in dry fermented sausages
(Berdagué et al., 1993; Ansorena et al., 1998) and Frankfurter sausages (Chevance and
Farmer, 1999). Berdagué et al. (1993) observed a significant influence of the nature of
the starter culture on the formation of 2,3-octanedione in dry sausages.
2,3-Octanedione has also been detected in fish. At first, the dione was only found in
some freshwater fish species but not in the surveyed saltwater species (Josephson et al.,
1984). Later on, Grimm et al. (2000) detected this substance in the headspace of cooked
catfish and Girard and Durance (2000) in the headspace of canned sockeye and pink
salmon in traces. The dione could also be identified in fish oil, such as rancid catfish oil
and crude menhaden oil (St. Angelo et al., 1987b) and coho salmon oil (Josephson et al.,
1991). Moreover, traces of 2,3-octanedione were present in fish sauce (Peralta et al.,
1996) and in fish oil enriched mayonnaise (Hartvigsen et al., 2000). Additionally, Cha
et al. (1992) identified it for the first time in crayfish.
Up to now, the origin of 2,3-octanedione is not fully understood. In many investigations
fatty acids or lipids in general are named as possible precursors and the formation of the
dione in connection with an oxidative process is reckoned. Drumm and Spanier (1991)
e.g. admitted that ketones such as 2,3-octanedione are products of lipid oxidation, but
that the mechanisms for their formation are not elucidated yet. Meynier et al. (1998,
1999) investigated the volatile compounds from oxidised muscle phospholipids of pork
and turkey but were not able to identify the structure of the precursor of 2,3-octanedione.
Literature Review
17
However, a strong correlation between the formation of 2,3-octanedione and of hexanal
was observed. This findings led the authors to suggest that the dione and hexanal are a
consequence of comparable or similar mechanisms involving n-6 fatty acid oxidation.
Studies by Suzuki (1985) on the influence of the diet on the flavour of lamb meat led to
the suggestion that 2,3-octanedione might be formed as a metabolite from a direct
precursor present in grass. Autoxidation of linoleic acid was suggested to be a possible
pathway. Young et al. (1997) investigated the effect of the diet on the volatiles of lamb
fat, and advanced a hypothesis that linked the formation of 2,3-octanedione to the
enzyme lipoxygenase and linolenic acid. It is also interesting to note that the formation
of 2,3-octanedione in pork during storage was reduced by Pseudomonas fragi (ChungWang et al., 1997). Taylor and Mottram (1990) identified 2,3-octanedione as an
oxidation product of methyl arachidonate, which was confirmed by Artz et al. (1993).
However, since arachidonic acid is of animal origin, this cannot explain the occurrence
of 2,3-octanedione in plants. Recently, Pompizzi et al. (2000) identified 2,3-octanedione
as a photooxidative degradation product of pentyl DiMeF. Starting from the
cycloaddition of singlet oxygen to DiMeF(9,5) to a bicyclic furan endoperoxide,
Pompizzi (1999) proposed two pathways with either an epoxy dioxoene or a furan
diepoxide as an intermediate (Fig. 4).
18
Literature Review
+ 1O2
R2
R1
O
R1 = (CH2)3CH3
R2 = (CH2)7COOCH3
R2
R1
O
O
O
O
O
R2
R1
R2
O
O O
H+
+H2O
HO
OH
H+
-H2O
R2
R1
O
R1
H
O O
+ 2 H2O
HO
HO
OH
OH
+
R1
O
R2
-2H
O
O
+
R1
O
R2
O
Figure 4: Proposed pathway for the photooxidative formation of 2,3-octanedione
(Pompizzi, 1999)
Reaction of the epoxide moiety with water leads to the dihydroxy compound which then
is oxidised to the corresponding diones. Reaction of the diepoxyde moiety with water
leads to the tetra diole, which again gives the dihydroxy compound under elimination of
water.
2.2.3
Bovolide and Dihydrobovolide
Bovolide and dihydrobovolide are two of the dimethyl substituted α,β-unsaturated-γlactones, which were initially used as artificial flavour additives in tobacco products
(Schumacher and Roberts, 1966). Some years later, dihydrobovolide (Kaneko and Mita,
1969) and bovolide (Demole and Berthet, 1972) were isolated from Burley tobacco
leaves. These findings were confirmed for example by investigations on the volatile
Literature Review
19
neutral fraction derived from Burley and Virginia tobacco (Forsblom et al., 1990).
The odour of bovolide is described as celery-like (Boldingh and Taylor, 1962;
Mookherjee and Wilson, 1990), fruity and pleasant (Takahashi et al., 1980), or celeryand lovage-like (Näf and Velluz, 2000). Dihydrobovolide possesses a characteristic
celery-like aroma as well (Sakata and Hashizume, 1973, Yamanishi et al, 1973;
Mookherjee and Wilson, 1990). The odour thresholds of bovolide and of dihydrobovolide are 100 pg/l and 480 pg/l in air and 2.0 µg/kg and 500 µg/kg in oil orthonasal,
respectively (Pompizzi, 1999).
Recently, studies by Kawakami (2002) revealed a slightly different aroma profile and
threshold level of the enantiomers (+)- and (-)-dihydrobovolide. The (+)-enantiomer
exhibits a metallic, spicy, green tea note, whereas the (-)-enantiomer develops a light and
spicy green tea note. The threshold level of (-)-dihydrobovolide was 1.6 ppm and lower
than that of (+)-dihydrobovolide (3.7 ppm).
Bovolide was first identified in butter by Boldingh and Taylor (1962) and Lardelli et al.
(1966) and was named to denote its bovine origin. Later on, the lactone was found in
various types of tea, such as green tea (Horita and Hara, 1984; 1985), semi fermented
Pouchong tea (Kawakami et al., 1986), pickled tea Miang (Kawakami et al., 1987), piled
tea Toyama Kurocha (Kawakami and Shibamoto, 1991), Oolong tea (Kawakami et al.,
1995) and black tea (e.g. Owuor and Obanda, 1999). Bovolide has been reported to
occur in peppermint oil (Takahashi et al., 1980), soy (Ames and MacLeod, 1984),
strawberry yam (Barron and Etiévant, 1990), essential oil of Pelargonium species
(Kayser et al., 1998), cooked spinach leaves (Näf and Velluz, 2000) and illuminated
samples of garden cress, woodruff, wheat germ oil and shrimps (Pompizzi et al., 2000).
Additionally, Bailey et al. (1994b) detected bovolide in lamb fat.
Dihydrobovolide has been reported to occur in the oil of Japanese peppermint (Sakata
and Hashizume, 1973), Ceylon flavoured tea (Yamanishi et al., 1973), cooked rice
(Yajima et al., 1978), leaves of Lycium chinense (Sannai et al., 1983), alfalfa (Kami,
1983), blended endive (Götz-Schmid and Schreier, 1986), the marine green alga species
Ulva pertusa (Fujimura et al., 1990), woodruff (Wörner and Schreier, 1991) and
illuminated dried mushrooms (Pompizzi et al., 2000). In dried bonito, the analogous
20
Literature Review
compound 2,3-dimethylhept-2-en-4-olide was also identified beside the dihydrobovolide (Yajima et al., 1983).
Both bovolides have been identified in green tea (e.g. Horita et al., 1985, Kawakami et
al., 1989), green mate and roasted mate (Kawakami and Kobayashi, 1991), the flower of
Michelia champaca (Kaiser, 1991), Rooibos tea (Kawakami et al., 1993) and dried
illuminated lamb‘s lettuce (Pompizzi et al., 2000). In animal samples, bovolide as well
as dihydrobovolide were found in cooked beef (MacLeod, 1991) and in shrimp powder
(Sarelse et al., 1994).
Boldingh and Taylor (1962) assumed that the bovolide found in butterfat originally
derived from the fodder. Based on the absence of other homologues they concluded that
the biosynthesis of bovolide is not related to normal fatty acid metabolism. Lardelli et
al. (1966) reported that by chromatographing grass extracts a fraction was obtained with
a distinct celery flavour. This was taken as an indication that bovolide is probably
present in grass. Further MacLeod (1991) assumed that the bovolide in beef originated
from grass as fodder. The results of the study by Bailey et al. (1994b) on the influence
of finishing diets on lamb flavour indicated an origin from grass as well. The authors
identified bovolide as one of the compounds having a strong positive relationship with
lamby flavour. Fat from animals finished on grain was significantly less lamby in flavour
than fat from animals finished on forage.
Horita et al. (1985) found the bovolides to be formed by light-induced chemical
reactions. However, in a study on the light-induced off-flavour of green tea, these
compounds turned out not to be responsible for the organoleptic sunlight flavour of
green tea (Horita, 1987).
Sarelse et al. (1994) proposed a pathway for the formation of bovolide by
photooxidative degradation of pentyl DiMeF (Fig. 5).
Literature Review
21
+ 1O2
R
O
O
R = (CH2)nCOOCH3
O
R
O
R1OH
n = 6 or 8
H+
O
OR1
R
OOH
R1
OH
∆ / H+
O
O
OR1
- R1OH
O
O
R1 = CH3 or H
Figure 5: Proposed pathway for the photooxidative formation of bovolide
(Sarelse et al., 1994)
Some years later, Pompizzi et al. (2000) confirmed the oxidative formation of bovolides
from pentyl DiMeF in model experiments and proposed pathways for the autoxidative
(Fig. 6) and for the photooxidative formation (Fig. 7) of bovolide. Recently, Kawakami
(2002) discussed an alternative way for the photooxidative formation of bovolide and
dihydrobovolide starting with a fatty acid (Fig. 8).
22
Literature Review
R1 = (CH2)3CH3
R2 = (CH2)7COOCH3
R2
R1
O
R
RH
R2
R1
R2
R1
.......
O
O
R
3O
2
RH
- OH
R1
R2
R1
O
OOH
- R2-CH2
O
O
Figure 6: Proposed pathway for the autoxidative formation of bovolide
(Pompizzi et al., 1997)
Literature Review
23
R2
R1
+ 1O 2
R2
R1
O
O
R1 = (CH2)3CH3
R2 = (CH2)7COOCH3
O
O
R1
R2
O
O
O
- R2CH2OH
R1
O
O
Figure 7: Proposed pathway for the photooxidative formation of bovolide
(Pompizzi et al., 2000)
fatty acid
O
hν
HO
O
HO
+ pyruvic acid
OH
HOOC
O
O
O
O
O
O
Figure 8: Hypothetical photooxidative formation of bovolide and dihydrobovolide
(Kawakami, 2002)
24
2.2.4
Literature Review
Pentanal
According to the general description the odour of pentanal is pungent and almond-like
(e.g. Schnabel et al., 1988). In addition, quite different odour perceptions were
associated with pentanal such as green, sweet and fatty (van Ruth et al., 1999) and
caramel, fruity and musty (van Ruth et al., 2000). Several odour thresholds for pentanal
measured in different media have been published (Tab. 4.).
Table 4: Odour thresholds for pentanal in different media
medium
threshold
reference
air
34 ng/l
Hall and Andersson (1983)
39 ng/l
Boelens and van Gemert (1987)
42 µg/kg (orthonasal)
Boelens and van Gemert (1987)
12 µg/kg (orthonasal)
Buttery et al. (1988)
20 µg/kg (orthonasal)
Schnabel et al. (1988)
76 µg/kg (retronasal)
Boelens and van Gemert (1987)
240 µg/kg (orthonasal)
Boelens and van Gemert (1987)
150 µg/kg (retronasal)
Boelens and van Gemert (1987)
267 µg/kg
Brewer and Vega (1995)
130 µg/kg
Boelens and van Gemert (1987)
water
oil
beef system
(meat/water)
milk
Pentanal is a common volatile aroma compound in food. Some published results are
worth to be mentioned in connection with the present work. Pentanal has been shown to
be the most abundant volatile in field-dried fescue hay (Mayland et al., 1997) and one of
the most abundant volatiles in cooked broccoli (Hansen et al., 1997). Hara (1989)
identified pentanal as one of the photochemically produced off-flavour components of
green tea. Bailey et al. (1994b) have investigated the influence of finishing diets on lamb
Literature Review
25
flavour. The fat from animals finished on grain exhibited a significantly less lamby
flavour compared to fat from animals finished on forage. Pentanal was part of the
volatile compounds which correlated significantly with the lamby flavour strength. St.
Angelo et al. (1987a) suggested to use pentanal as a marker to follow the development
of WOF.
Pentanal is known to be a minor oxidation product of certain fatty acids. This aldehyde
was identified as a secondary reaction product of the oxidation of linoleic acid (Forss,
1973; Yoshino et al., 1991; Frankel, 1998; Spiteller, 1998), of linolenic acid (Yoshino et
al., 1991), of arachidonic acid (Forss, 1973; Taylor and Mottram, 1990; Yoshino et al.,
1991; Artz et al., 1993), of palmitoleic acid (Forss, 1973), of oleic acid (Neff et al., 2000)
and of docosahexaenoic acid (Yoshino et al., 1991). Lardelli et al. (1966) observed a
formation of pentanal by oxidation of bovolide. Recently, Pompizzi et al. (2000)
identified pentanal as a photooxidative degradation product of pentyl DiMeF.
2.2.5
2,3-Butanedione
2,3-Butanedione (diacetyl) is known as a key aroma compound in butter and, in fact, its
generally accepted odour descriptor is buttery (Widder, 1994; Belitz et al., 2001). The
thresholds have been investigated in many studies in different media and a compilation
is given in Rychlik et al. (1998). Siek et al. (1969) e.g. obtained taste thresholds of
5.4 µg/l in water, 55 µg/l in oil, 14 µg/l in milk and 32 µg/kg in butter.
The dione is a potent aroma compound in milk-products such as yoghurt and cheese, but
also in bread, cooked fish, coffee and French fries. In beer and in soy bean-oil, 2,3butanedione contributes to the off-flavour (Belitz et al., 2001).
Several possible pathways of formation of 2,3-butanedione have been shown. In dairy
products, diacetyl is the result of the action of lactic acid bacteria and is biosynthesised
from citric acid (Gatfield, 1986). Artz et al. (1993) identified 2,3-butanedione to be a
decomposition product from oxidised methyl arachidonate. Hollnagel and Kroh (1998)
have shown in model experiments that the dione was a reaction product from nonenzymatic browning of D-glucose, D-fructose, maltose and maltulose. Finally, 2,3butanedione was identified as a photooxidative degradation product of pentyl DiMeF by
Pompizzi et al. (2000). However, with the exception of dairy products, the origin of
26
Literature Review
diacetyl could not be explained for all food products. For example the origin of 2,3butanedione as potent odorant in boiled trout is still unknown (Milo and Grosch, 1993).
27
3
EXPERIMENTAL PART
3.1
Material
3.1.1
Sample material
Dried samples of tarragon (Artemisia dracunculus), basil (Ocimum basilicum), savory
(Satureja hortensis), chervil (Anthriscus cerefolium), dill (Anethum graveolens), chive
(Allium schoenoprasum), onion (Allium cepi) and leek (Allium porrum) were obtained
in paper bags from J. Carl Fridlin Gewürze AG, Hünenberg, CH. Green tea samples
(Sencha, Fuji region Japan) were obtained in light-protected vacuum-bags from Peter
Oppliger AG, Lucerne, CH. The food samples were stored in light-protected vacuumbags at 4 °C before analysis.
3.1.2
Reference aroma compounds
2,3-Dimethylnona-2,4-dien-4-olide (bovolide) (Pompizzi, 1999)
2,3-Butanedione, Fluka 31530 (Fluka AG, Buchs, CH)
2,3-Dimethylnon-2-en-4-olide (dihydrobovolide) (Pompizzi, 1999)
3-Methyl-2,4-nonanedione (MND) (see chapter 3.2.1)
2,3-Octanedione (see chapter 3.2.2)
Pentanal, Fluka 94512
3.1.3
Model mixture of reference aroma compounds
A stock solution of the model mixture was prepared by dissolving 20 to 30 mg of each
of the aroma compounds pentanal, 2,3-butanedione, 2,3-octandione, MND, bovolide
and dihydrobovolide in 20 ml diethyl ether. A diluted aroma solution was prepared by
28
Experimental Part
diluting 1 ml of the stock solution to 10 ml with diethyl ether. In Tab. 5 the exact
concentrations of the components of the stock solution are shown.
Table 5: Concentrations of the components of the model mixture in the stock solution
aroma compound
concentration [g/l]
pentanal
1.0
2,3-butanedione
1.5
2,3-octanedione
1.4
MND
0.9
bovolide
1.6
dihydrobovolide
1.1
3.1.4
Dimethyl furan fatty acid standards
DiMeF(7,5)methyl ester, DiMeF(8,5)methyl ester, DiMeF(9,5)methyl ester,
DiMeF(11,5)methyl ester, DiMeF(11,3)methyl ester (Pompizzi, 1999).
3.1.5
Chemicals
All chemicals were purchased from J.T. Baker B.V. (Deventer, NL), Fluka AG (Buchs,
CH), Lancaster Synthesis GmbH (Frankfurt a. Main, D), Merck Ltd. (Darmstadt, D),
Riedel-de Haën Laborchemikalien GmbH & Co KG (Seelze, D), Separtis GmbH
(Grenzach-Wyhlen, D) or Siegfried AG (Synopharm, Basel, CH) and were of analytical
quality.
Acetonitrile (Fluka 00700), boron trifluoride-methanol 10 % (Fluka 15716), carbon
tetrachloride (Fluka 87031), copper(II)acetate (Riedel-de Haën 25038), dichloromethane (Baker 7053), diethyl ether (Riedel-de Haën 32203), 2,6-di-tert-butyl-phydroxy-toluene (BHT) (Fluka 34750), ethanol (Fluka 02860), ethyl decanoate (Fluka
21430), ethyl valerate (Fluka 94540), hexane (Fluka 52760), iodomethane
Experimental Part
29
(Fluka 67690), isolute HM-N (Separtis 9800-0060), isopropanol (Riedel-de Haën
27225), magnesium sulfate anhydrous (Fluka 63136), meso-tetraphenyl porphyrine
(Fluka 88071), methanol absolute (Fluka 65543), methanol (Baker 8045), methylene
blue (Fluka 66720), methyl undecanoate (Fluka 94118), 2,4-nonanedione (Lancaster
1151), 2-octine (Fluka 74972), potassium hydroxide (Siegfried 16200-06),
ruthenium(IV)oxide (Fluka 84065), silica gel 60 (Fluka 60752), sodium (Fluka 71172),
sodium chloride (Merck 6400), sodium hydrogen carbonate (Fluka 71628), sodium
periodate (Fluka 71862), sulfuric acid (Merck 100731).
3.2
Synthesis of reference aroma compounds
3.2.1
3-Methyl-2,4-nonanedione
MND was synthesised according to Guth and Grosch (1989). A solution of sodium
ethylate was prepared by dropwise addition of 9 ml ethanol to 0.4 g (16 mmol) sodium
under stirring. After complete dissolution of the sodium, the solution was heated to
70 °C and treated dropwise under stirring with 2.8 g (16 mmol) 2,4-nonanedione and
1.1 ml (16 mmol) iodomethane. The mixture was refluxed at 75 °C for 4 h, treated with
additional 0.5 ml (8 mmol) iodomethane and refluxed at 80 °C for 1 h. Most of the
ethanol was then distilled off and the residue poured in 100 ml ice water. After extraction
of the mixture with diethyl ether (3 x 150 ml), the combined ethereal extracts were
washed with brine, dried (MgSO4) and concentrated under reduced pressure. The yield
of crude MND, obtained as a yellow oil, was 3.2 g (102 %) with a purity of 81 % (GCFID).
The purification was performed according to Adams and Hauser (1944). To 0.6 g of
crude MND, 30 ml of a hot 7 % (w/v) copper(II)acetate solution was added and the
mixture refluxed under stirring at 80 °C for 10 min. After cooling to 0 °C, the mixture
was filtered, the residue (copper salt) washed with ice-cold methanol (2 x 10 ml) and
recrystallised from boiling methanol. The whitish crystals were dissolved in 10 ml of a
10 % (w/v) sulfuric acid solution and the solution extracted with diethyl ether (3 x
20 ml). The combined ether extracts were washed with brine to neutral, dried (MgSO4)
and concentrated under reduced pressure. The yield of MND, obtained as pale yellow
oil, was 0.29 g (48 %) with a purity of 98 % (GC-FID). The analytical data were in
agreement with those reported by Pompizzi (1999).
30
3.2.2
Experimental Part
2,3-Octanedione
According to Zibuck and Seebach (1988), 39.8 g (186.1 mmol) sodium periodate was
added to a mixture of 5 g (45.5 mmol) 2-octine in 280 ml water, 200 ml acetonitrile and
200 ml carbon tetrachloride. The mixture was stirred vigorously at room temperature
until two clear phases resulted. Then 133 mg (0.8 mmol) ruthenium(IV)oxide was added
under ice cooling. The mixture was stirred at room temperature for 30 min. After
addition of 300 ml water, the organic phase was separated, the aqueous phase extracted
with dichloromethane (3 x 100 ml), the combined organic phase dried (MgSO4) and
concentrated by means of a Vigreux column. The residue (2.19 g, 33.9 %) was purified
by chromatography (silica gel 60, l = 40 cm, d = 7 cm, hexane/dichloromethane = 1:1
(v/v)), distilled at reduced pressure in a Kugelrohr to yield 1.55 g (70 %) 2,3octanedione as pale yellow oil with a purity of 97 % (GC-FID). The analytical data were
in agreement with those reported by Pompizzi (1999).
3.3
Analytical methods
3.3.1
Capillary gas chromatography (GC-FID)
GC was performed using the on column (OC) injection technique on a Trace GC 2000
series gas chromatograph (ThermoQuest CE Instruments Ltd., Milano, I) with flame
ionisation detector (FID). A polar fused silica SW-10 column (60 m, 320 µm ID,
0.25 µm film thickness, Supelco Inc., Bellefonte, USA) with a deactivated fused silica
pre-column (2.8 m, 530 µm ID, J & W Scientific Inc., Folsom, USA) was used. Data
processing was achieved with ChromCard, version 1.06 software (ThermoQuest CE
Instruments). Tab. 6 shows the experimental conditions for GC analysis. For semiquantitative analysis, the peak area ratio (RFID) to an internal standard (ISTD) was
determined according to equation 1.
Experimental Part
31
Table 6: Experimental conditions for GC-FID analysis
carrier gas
He
carrier flow [ml/min]
1.8
injection mode
OC
injection temperature [°C]
20
injection volume [µl]
13
detection temperature [°C]
240
temperature programming
isothermal stage 1 [°C]; [min]
40; 18
rate 1 [°C/min]
6
isothermal stage 2 [°C]; [min]
220; 15
AX
R FID = ----------------A ISTD
where:
(equation 1)
RFID: relative amount of compound X as compared to the
internal standard
AX:
peak area of compound X
AISTD: peak area of internal standard
3.3.2
Capillary gas chromatography-mass spectrometry
(GC-MS)
GC-MS was performed using the OC injection technique on a GC 8065 gas
chromatograph (Fisons Instruments Inc., Milano, I) directly coupled to a SSQ 710 mass
spectrometer (Finnigan Inc., San Jose, USA). A polar fused silica SW-10 column (60 m,
320 µm ID, 0.25 µm film thickness, Supelco) with a deactivated fused silica pre-column
32
Experimental Part
(2.8 m, 530 µm ID, J & W Scientific) was used. Data processing was achieved with the
ICIS 2 version 8 software (Finnigan). Tab. 7 shows the three different sets of conditions
for GC-MS analysis. Semi-quantitative analysis was performed by evaluation of the
peak area ratio (RMS) to an internal standard (see also equation 1) based either on the
total ion current (TIC) or the characteristic fragment ions at m/z 58 (pentanal), 74
(methyl undecanoate), 83 (dihydrobovolide), 86 (2,3-butanedione), 88 (3-hydroxy-3methyl-2,4-nonanedione (HMND), ethyl decanoate, ethyl valerate), 99 (2,3-octanedione, MND) and 124 (bovolide).
.
Table 7: Experimental conditions for GC-MS analysis
method
A
B
C
carrier gas
He
He
He
carrier flow [kPa]
100
100
100
injection mode
OC
OC
OC
injection temperature [°C]
20
20
20
injection volume [µl]
10
1
1
isothermal stage 1 [°C]; [min]
40; 12
50; 5
90; 5
rate 1 [°C/min]
6
10
10
isothermal stage 2 [°C]; [min]
220; 30
240; 10
240; 10
ionisation potential [eV]
70
70
70
ion source temperature [°C]
150
150
150
manifold temperature [°C]
70
70
70
interface temperature [°C]
200
200
200
mass range [amu]
40-420
40-420
40-420
scan time [s]
1
1
1
temperature programming
Experimental Part
3.3.3
33
Capillary gas chromatography-ion trap mass spectrometry
(GC-MS/MS)
GC-MS/MS was performed either on a Trace GC 2000 series gas chromatograph
(ThermoQuest CE Instruments) directly coupled to a GCQ Plus mass spectrometer
(ThermoQuest Finnigan Inc, San Jose, USA) using an apolar fused silica column Rtx5MS (30 m, 250 µm ID, 0.25 µm film thickness, Restek Corp., Bellefonte, USA) or on
a Varian 3800 gas chromatograph (Varian Inc., Palo Alto, USA) directly coupled to a
Varian Saturn 2000 mass spectrometer (Varian) using an apolar fused silica column
DB5-MS (28 m, 250 µm ID, 0.25 µm film thickness, J & W Scientific). The
experimental conditions for the GC-MS/MS analysis and the MS/MS ion preparation are
listed in Tab. 8 and Tab. 9.
Table 8: Experimental conditions for GC-MS/MS analysis
method
D
E
facility
ThermoQuest
Varian
carrier gas
He
He
carrier flow [ml/min]
1.6
1.6
injection mode
splitless 1.0 min
splitless 0.5 min
injection temperature [°C]
250
250
injection volume [µl]
1
2
isothermal stage 1 [°C]; [min]
60; 1
60; 1
rate 1 [°C/min]
10
10
isothermal stage 2 [°C]; [min]
300;10
300;10
ionisation potential [eV]
70
70
ion source temperature [°C]
150
150
manifold temperature [°C]
70
70
interface temperature [°C]
275
250
temperature programming
34
Experimental Part
Data processing was achieved with the XCalibur version 1.1 software (ThermoQuest
Finnigan) or with the Saturn View (TM) version 5.41 software (Varian), respectively.
Samples were injected in the splitless injection mode.
For quantification, a six or eight point calibration curve (see chapter 4.1.1) was
established and for semi-quantitative analysis, the peak area ratio (RMS/MS) to an
internal standard was determined (see also equation 1). For both calculations the
characteristic product ions at m/z 123 (pentyl DiMeF) and 109 (propyl DiMeF) were
used.
Table 9: Experimental conditions for the MS/MS ion preparation
method
D
E
179 (pentyl DiMeF)
179 (pentyl DiMeF)
151 (propyl DiMeF)
151 (propyl DiMeF)
range [amu]
1
1
isolation time [ms]
12
10
collision energy [V]
1.2
1.2
collision time [ms]
15
12
qz value
0.45
0.45
isolation parameters:
precursor ion [amu]
dissociation parameters:
3.3.4
Capillary gas chromatography-infrared spectrometry
(GC-IR)
GC-IR was performed using the OC injection technique on a HP 5890 gas
chromatograph (Hewlett Packard Co, Palo Alto, USA) with a HP 5965 IR-detector
(Hewlett Packard). A medium-polar fused silica DB 1701 column (10 m, 180 µm ID,
0.4 µm film thickness, J & W Scientific) was used. Data processing was achieved with
the GRAMS/32 software (Portmann Instruments AG, Biel-Benken, CH). Tab. 10 shows
the experimental conditions for GC-IR analysis.
Experimental Part
35
Table 10: Experimental conditions for GC-IR analysis
carrier gas
He
carrier flow [kPa]
100
injection mode
OC
injection temperature [°C]
20
injection volume [µl]
2
temperature programming
isothermal stage 1 [°C]; [min]
40; 1
rate 1 [°C/min]
10
isothermal stage 2 [°C]; [min]
300; 10
3.3.5
Capillary gas chromatography-olfactometry (GC-O)
For sniffing experiments, a GC-FID system of the type HP GC 5890 series II (Hewlett
Packard) was equipped with a column end split, leading to a sniffing port for
olfactometry. A polar fused silica SW-10 column (60 m, 320 µm ID, 0.25 µm film
thickness, Supelco) was used. Data processing was achieved with a HP 3365 Series II
integrator (Hewlett Packard) and the Chem Station Version A.03.34 software (Hewlett
Packard). Samples were injected in the split injection mode. Tab. 11 shows the
experimental conditions for GC-O analysis.
36
Experimental Part
Table 11: Experimental conditions for GC-O analysis
carrier gas
He
carrier flow [kPa]
100
injection mode
split 1:12
injection temperature [°C]
240
injection volume [µl]
2
detection temperature [°C]
240
temperature programming
isothermal stage 1 [°C]; [min]
90; 7
rate 1 [°C/min]
20
isothermal stage 2 [°C]; [min]
150; 10
rate 2 [°C/min]
20
isothermal stage 3 [°C]; [min]
240; 10
3.4
Extraction methods
3.4.1
Simultaneous distillation solvent extraction (SDE)
The extraction of the volatile compounds was performed in a micro steam distillation
apparatus (Chrompack Inc., Middelburg, NL) modified for solvents lighter than water
according to the design of Godefroot et al. (1981). 170 ml water, 200 µl of the diluted
aroma solution (see chapter 3.1.3) or 10.0 g of sample material and 21 µg ethyl
decanoate as internal standard (ISTD 1) were placed in a 250 ml roundbottom flask. For
herbs and vegetables 150 ml water, 6.0 g sample material and 12 µg ISTD 1 were used.
In a 5 ml pear-shaped flask 2 ml diethyl ether and 20 µg ethyl valerate as internal
standard (ISTD 2) were placed. The aqueous mixture was heated to boiling and the
organic solvent containing flask was heated in an oil bath set at 80 °C. The condenser
was cooled to -17 °C and micro-SDE carried out for 60 min after the water vapour
reached the condenser. After removing the heating sources, the solvent in the separation
chamber was transferred to the extract, 21 µg methyl undecanoate as internal standard
Experimental Part
37
(ISTD 3) were added and the extract dried (MgSO4). The samples were analysed with
GC-FID (Tab. 6) and/or GC-MS (Tab. 7, method A). The compounds were identified by
comparison of mass spectra and retention indices (RI, calculated according to Van den
Dool and Kratz (1962)) with reference substances. RFID and RMS were calculated with
either ISTD 1 or ISTD 3 as internal standards. To investigate a possible artefact
formation, a solution of 110 µg DiMeF(7,5)methyl ester, 110 µg DiMeF(9,5)methyl
ester and 160 µg DiMeF(11,5)methyl ester dissolved in 100 µl diethyl ether was
subjected to the same procedure.
3.4.2
Accelerated solvent extraction (ASE)
For the analysis of the FFA, the lipid fraction was extracted by accelerated solvent
extraction (ASE) using an ASE 200 equipment (Dionex Corp. Sunnyvale, USA). The
samples were finely ground immediately before extraction. To prevent oxidation during
the extraction, the solvent was degassed before use and 0.1 % BHT (w/v) was added.
The extraction conditions are listed in Tab. 12. The extract was dried (MgSO4) and
concentrated at reduced pressure.
The crude lipids were saponified under argon with 25 ml 0,5 M potassium hydroxide in
methanol for 15 min at reflux and then converted to methyl esters by refluxing under
argon with 20 ml 10 % (1.3 M) boron trifluoride in methanol for 5 min. After addition
of 100 ml satd. sodium hydrogen carbonate solution, 40 ml hexane and 280 µg
DiMeF(8,5)methyl ester as internal standard, the mixture was stirred for 10 min at room
temperature and extracted with hexane (5 x 40 ml). The organic phase was washed with
brine (2 x 40 ml), dried (MgSO4) and concentrated at reduced pressure. The residue was
stored under argon at -28 °C before analysis. For the GC-MS/MS analysis (for
quantification: method D (Tab. 8 and 9); for semi-quantitative analysis: method E
(Tab. 8 and 9)), aliquots were dissolved in hexane. The compounds were identified by
comparison of mass spectra, characteristic product ions and retention times with DiMeF
standards. Quantification and calculation of RMS/MS were performed with
DiMeF(8,5)methyl ester as internal standard.
38
Experimental Part
Table 12: Operating parameter for the ASE 200 extraction
sample amount [g]
approx. 10
hydromatrix [g]
approx. 2 (isolute HM-N)
pressure [MPa]
6,7
temperature [°C]
125
solvent [v/v]
hexane-isopropanol (3:2)
heating up phase [min]
6
static time [min]
10
static cycles
2
flush volume [ % of cell volume]
60
nitrogen flush [min]
3
extraction steps per sample
4
3.5
Light exposure experiments
3.5.1
Sample preparation
The dried samples were packed under air or oxygen atmosphere in transparent PE-film
bags and exposed to light (see chapter 3.5.2 and chapter 3.5.3) at room temperature for
different time periods. Samples before light exposure or stored in darkness under the
same conditions were used as reference.
3.5.2
Light exposure model system I
The experiments were carried out in a cardboard box (length = 115 cm, width = 47 cm,
height = 90 cm) with two fluorescent tubes (BIOLUX 36W/72-965, Osram AG,
Winterthur, CH) with an emission spectrum similar to that of the sun (Fig. 9). The
samples were placed at a distance of 45 cm from the light source. The illuminance was
adjustable from 1100 lx to 4500 lx. A regulation system with a photo cell (Reiter, 2000)
Experimental Part
39
Intensity [300 mW/1000 lm · 10 nm]
ensured a constant illuminance during the exposure period.
400
500
600
700 nm
Figure 9: Emission spectrum of the BIOLUX lamps (Osram, 1995/1996a)
3.5.3
Light exposure model system II
The experiments were carried out in a dark room. The light source consisted of two
sodium vapour-high pressure lamps (Vialox NAV E 140 de Luxe, Osram) placed at a
distance of 30 cm from the samples. The illuminance corresponded to 40‘000 lx. The
emission spectrum of the lamps was similar to that of the sun (Fig. 10).
Experimental Part
Intensity [300 mW/1000 lm · 10 nm]
40
400
500
600
700 nm
Figure 10: Emission spectrum of the Vialox lamps (Osram, 1995/1996b)
3.6
Oxidation experiments
The experiments were performed under photooxidative conditions at 4 °C in two
different organic solvents according to Pompizzi (1999). A special three neck glass
vessel (d1 = 4 cm, d2 = 10 cm, h = 14 cm) with a fritted glass bottom (DEMA 13/21,
Hans Mangels, Bornheim, D) was used. The light source consisted of two sodium
vapour-high pressure lamps (Vialox NAV E 150 de Luxe, Osram) with an illuminance
of 20‘000 lx each placed at a distance of 0.45 m from the reaction vessel.
Experiments with MND: 34 µg (0.17 mmol) MND, 144 µg ethyl decanoate (internal
standard) and 69 µg ethyl valerate (internal standard).
Experiments with 2,3-octanedione: 28 µg (0.2 mmol) 2,3-octanedione, 151 µg ethyl
decanoate (internal standard) and 52 µg ethyl valerate (internal standard).
The oxidation products were identified by comparison of RI and of the mass spectra by
spiking with reference substances. If reference substances were not available,
identification was based on mass spectral characteristics. The main degradation products
were analysed semi-quantitatively (RMS based on TIC) with the internal standard ethyl
decanoate.
Experimental Part
3.6.1
41
Conditions in hexane
In 200 ml hexane, 20 mg meso-tetraphenyl porphyrine was dissolved with the aid of an
ultrasonic bath and the dione and the two internal standards were added. The solution
was illuminated for 24 h under slightly bubbling oxygen. After 24 h, 0.6 mg BHT were
added, and the solution was concentrated to 3 ml at reduced pressure (50 - 60 mbar) by
means of a Vigreux column and ice cooling.
The same procedure was carried out in absence of light (dark oxidation), absence of
sensibilisator (unsensitised oxidation) and in absence of sensibilisator and argon instead
of oxygen (photochemical reaction).
The samples were stored at -80 °C before GC-MS analysis with method C (Tab. 7).
3.6.2
Conditions in methanol
A solution of the dione, the two internal standards and 30 mg methylene blue in 200 ml
methanol was illuminated for 24 h under slightly bubbling oxygen. After 24 h, 0.6 mg
BHT were added, and the solution was concentrated to 3 ml at reduced pressure
(35 mbar) by means of a Vigreux column and ice cooling. The residue was diluted with
10 ml water and the mixture extracted with diethyl ether (3 x 10 ml). The combined
organic phase was dried (MgSO4) and concentrated to 3 ml at 120 mbar by means of a
Vigreux column and ice cooling.
The same procedure was carried out in absence of light (dark oxidation). The samples
were stored at -80 °C before GC-MS analysis with method B (Tab. 7).
42
43
4
RESULTS AND DISCUSSION
4.1
Analysis of furan fatty acids
4.1.1
Development of a method using ion trap gas chromatography-mass spectrometry
In order to develop an appropriate GC-MS/MS method for the analysis of FFA two
points had to be clarified. At first, the main diagnostic ion of each class of FFA (pentyl
DiMeF and propyl DiMeF) was determined. Secondly, the conditions to generate these
diagnostic ions in preferably high intensity were optimised. The objective was to attain
a satisfactory sensitivity even in complex samples.
DiMeF show analogous fragmentation with allylic cleavage of the carboxyl side chains
(Wahl, 1994). The resulting base peak is therefore only dependent on the alkyl side chain
with m/z 179 for the pentyl DiMeF and m/z 151 for the propyl DiMeF. These
characteristic fragment ions were chosen for further fragmentation. MS/MS experiments
of the precursor ion at m/z 179 resulted in a characteristic base peak at m/z 123 by allylic
cleavage of the pentyl side chain (Fig. 11 A). The peak at m/z 123 was chosen as
diagnostic ion for the analysis of pentyl DiMeF. Fragmentation of the precursor ion for
propyl DiMeF at m/z 151 surprisingly led to a characteristic base peak at m/z 109
(Fig. 11 B). It is assumed that a vinylic cleavage of the propyl side chain dominated over
the allylic cleavage at the MS/MS conditions used in this study. The peak at m/z 109 was
therefore chosen as diagnostic ion for the analysis of propyl DiMeF.
44
Results and Discussion
123
100
+
80
179
109
60
O
40
CH2
123
+H
110
20
A
0
100
110
120
130
140
150
m/z
160
170
180
190
200
109
100
+
80
60
CH2
O
122
40
151
109
+H
20
123
B
0
100
110
120
130
140
150
m/z
160
170
180
190
200
Figure 11: MS/MS spectrum of characteristic precursor ions for pentyl and propyl
dimethyl furan fatty acids
A: m/z 179
B: m/z 151
As an example for the efficiency of the method, the ion trap GC-MS chromatograms of
the fatty acid methyl ester extract of chervil are shown in Fig. 12. In the GC-MS analysis
(Fig. 12 A), the FFA either co-eluted with other components present in the extract or
could not be detected. With GC-MS/MS (Fig. 12 B), different pentyl DiMeF were
separated in the product ion trace of the diagnostic ion at m/z 123.
Results and Discussion
45
100
80
60
40
20
A
0
100
DiMeF(11,5)
80
60
DiMeF(8,5)
40
20
DiMeF(9,5)
B
0
12
14
16
18
Time (min)
20
22
24
Figure 12: Ion trap GC-MS chromatograms of fatty acid methyl ester extract of chervil
(A) MS chromatogram, based on full scan (m/z 40-450)
(B) MS/MS chromatogram, based on the diagnostic product ion (m/z 123)
Grabic et al. (2000) stated that at a constant isolation time and excitation time the yield
of product ions from an MS/MS experiment depends on the collision energy added to the
precursor ions. The evolution of intensity of the product ion as a function of the
excitation voltage was tested at a constant qz value = 0.45, isolation time of 12 ms and
excitation time of 15 ms. The analysis was performed in two steps. Firstly the peak area
of the chosen diagnostic ion was determined with excitation voltages between 0 and 2 V.
Secondly the excitation voltage was varied in the area of the higher intensity. In addition
to a high peak area of the product ion, incomplete fragmentation of the precursor ion was
a matter of concern as well. The relationship between collision energy and yield of the
diagnostic ions for pentyl DiMeF and propyl DiMeF was established with DiMeF(9,5)
and DiMeF(11,3) standards, respectively (Tab. 13 and Tab. 14).
46
Results and Discussion
Table 13: Intensity of the diagnostic ion for pentyl DiMeF at varying collision energy
excitation voltage
peak area diagnostic ion
precursor ion available
[V]
(m/z 123)
(m/z 179)
0
-
yes
1.0
10‘775‘745
yes
2.0
2‘763‘032
no
0.9
10‘371‘469
yes
1.2
11‘853‘346
yes
1.5
4‘729‘902
yes
Table 14: Intensity of the diagnostic ion for propyl DiMeF at varying collision energy
excitation voltage
peak area diagnostic ion
precursor ion available
[V]
(m/z 109)
(m/z 151)
0
-
yes
0.9
40‘788‘468
yes
1.2
50‘608‘840
yes
1.5
13‘874‘301
no
0.9
52‘525‘853
yes
1.0
54‘365‘911
yes
1.2
55‘019‘434
yes
Results and Discussion
47
The highest intensity of the target product ions was obtained at an excitation voltage of
1.2 V for both the pentyl DiMeF and the propyl DiMeF. At low excitation voltages the
fragmentation of the precursor ions was too low. On the other hand, at high excitation
voltages too many low-molecular weight product ions were formed, which were not hold
back in the ion trap at the chosen qz value. Duplication of the excitation time to 30 ms
did not enhance the intensity of the diagnostic ions (data not shown). Therefore a
possible effect of the excitation time as well as the isolation time was supposed to be
negligible and was not further studied. Grabic et al. (2000) have not observed a
significant improvement in sensitivity at different values of excitation time and isolation
time in the analysis of PCDDs and PCDFs. However, the authors obtained a higher
response of the product ion at an increased collision pressure compared to a lower one.
It is likely that the sensitivity in the analysis of FFA could be enhanced as well by
optimising the pressure of the collision gas.
Based on the expected allylic cleavage of the alkyl and carboxyl side chains (Fig. 13),
the precursor ions and diagnostic product ions of different classes of FFA were deduced.
This allowed a qualitative screening of homologous mono- and dimethyl substituted
FFA in the samples without using standards for verification. As discussed for propyl
DiMeF, vinylic cleavage of the alkyl side chain can dominate the allylic cleavage. In this
case, qualitative analysis using the predicted diagnostic ion is still possible, however,
with a decreased sensitivity. For identification of the FFA, the corresponding molecular
ion was taken as an additional criterion. The second fragmentation of some components
present in the methyl ester extracts, however, led to ions with m/z values identical to the
suggested diagnostic product ions of the FFA. In these cases, the complete MS/MS
fragmentation spectra were interpreted to differentiate peaks not derived from FFA.
48
Results and Discussion
precursor ion
R2
H3C(H2C)n-1
+H
R1
O
+H
(CH2)m-1COOCH3
R1 = CH3
R2 = H; CH3
n=2-6
m = 2 - 14
product ion
Figure 13: Precursor ion and product ion obtained by allylic cleavage of furan fatty acid
methyl esters
In the samples, DiMeF(9,5) and DiMeF(11,5) as well as DiMeF(11,3) were quantified
with the internal standard method. DiMeF(8,5) was used as ISTD since DiMeF with an
odd number of carbon atoms have not been reported to occur in plants yet. Because of
the wide range of concentrations of DiMeF(11,5) in the different samples ranging from
0.5 mg/kg to over 300 mg/kg, two separate calibration curves were established. For the
other two FFA, calibration curves were established in the range of the expected
concentrations in the samples. Mean values of independent duplicates were used to
calculate the calibration graphs (Fig. 14).
Results and Discussion
Figure 14: Calibration graphs for quantification of different furan fatty acids (n=2)
A: DiMeF(9,5)
B: DiMeF(11,3)
C: DiMeF(11,5) high concentrations
D: DiMeF(11,5) low concentrations
49
50
Results and Discussion
Several methods for the quantitative determination of FFA have been published. In these
methods the lipids have to be pre-fractionated to obtain a FFA-enriched fraction (see
Stansby et al., 1990 and the literature cited therein). Besides the fact that all these
methods are time-consuming, the risk for losses or formation of artefacts have to be
considered. Recently, methods for the direct identification or determination of FFA have
been reported. They require, however, the use of unusual procedures and facilities such
as multidimensional GC-MS (Wahl et al., 1995) or HPLC-GC with a FID-PID (photo
ionisation detector) system (Boselli et al., 2000). The advantages of the ion trap GCMS/MS technique as described in the present work are manifold. It allows the
determination of FFA in the methyl ester extract of plant lipids in the presence of coeluates without prior separation, and it is fast and sensitive. The method using GCMS/MS has successfully been applied to determine DiMeF in green tea and in dried
herbs and vegetables (see chapter 4.1.2).
4.1.2
Furan fatty acid content of green tea and dried green herbs
and vegetables
The contents of FFA in dried green plant material was determined as described in
chapter 3.3.3 and chapter 3.4.2. Leek, chervil, dill and green tea were analysed in
independent duplicates, denoted A and B. The amounts of FFA in the investigated
samples are shown in Tab. 15. Within the group of pentyl FFA, two monomethyl
substituted, two dimethyl substituted and two olefinic derivatives were identified in at
least some of the samples. One propyl FFA was found exclusively in chive. The samples
were also screened for FFA with a butyl, hexyl or heptyl side chain, based on the
considerations in chapter 4.1.1. None of them were identified in any sample. The
dimethyl substituted FFA were analysed quantitatively whereas the monomethyl
substituted FFA could only be analysed qualitatively because of lack of standard
substances. MeF(9,5) was found in green tea, dill and leek, whereas MeF(11,5) was
present in green tea, chervil and chive. To our knowledge no data on the FFA
composition of tarragon, basil, savory, chervil, dill, onion and leek are available in the
literature.
The amounts of the pentyl DiMeF differed considerably in the investigated herbs and
vegetables. In general, DiMeF(11,5) was present in higher amounts compared to
DiMeF(9,5), which is similar to the findings by Hannemann et al. (1989).
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.7
n.d.
n.d.
n.d.
n.d.
n.d.
basil
savory
chervil A
chervil B
dill A
dill B
chive
onion
leek A
leek B
green tea A
green tea B
µg/g dry matter
peak area ratio to DiMeF(11,5) in %
not detected
traces
detected
n.d.3
tarragon
1.
2.
3.
4.
5.
DiMeF(11,3)1
sample
d.
d.
d.
d.
n.d.
n.d.
d.
d.
n.d.
n.d.
n.d.
n.d.
n.d.
d.
d.
n.d.
n.d.
n.d.
d.
n.d.
n.d.
9.5
6.7
n.d.
n.d.
n.d.
tr.
3.6
tr.
7.1
8.4
d.5
d.
n.d.
n.d.
0.6
DiMeF(9,5)1
n.d.
n.d.
n.d.
MeF(9,5) MeF(11,5)
335.3
241.9
32.9
28.1
0.6
18.9
11.5
35.2
107.8
97.1
2.5
7.5
111.1
DiMeF(11,5)1
Table 15: Concentrations of furan fatty acids in green tea, different herbs and vegetables
2.0
1.4
tr.
tr.
tr.
5.2
n.d.
25.9
13.4
7.6
2.7
1.7
tr.
tr.
tr.
7.1
n.d.
23.5
14.8
13.5
n.d.
tr.
tr.4
tr.
4.3
DiMeF(11:1, 5)2
5.1
DiMeF(11, 5:1)2
Results and Discussion
51
52
Results and Discussion
DiMeF(11,5) was found in all samples, whereas DiMeF(9,5) could not be detected in
savory, basil, leek and onion. Chervil and tarragon were shown to be rich (> 100 µg/g)
in DiMeF(11,5), whereas in onion very small amounts (< 1 µg/g) of this FFA were
detected. These findings are in agreement with those of Hannemann et al. (1989), who
claimed that FFA occur in higher amounts in the green parts of plants than in the trunks,
roots and seeds. However, the values obtained here were approximately ten times lower
when compared to results published for dried parsley (Masanetz and Grosch, 1998) and
dried spinach (Masanetz et al., 1998).
Chive was shown to contain 18.9 µg/g DiMeF(11,5) which agrees quite well with the
16 µg/g found by Hannemann et al. (1989). The content of DiMeF(11,3) in chive was
very low (1.7 µg/g); Hannemann et al. (1989) detected this propyl DiMeF in small
amounts (< 4 µg/g) in grasses (blade), clover, birch (leaf), wheat and potato (leaf), but
not in chive. In general, FFA with a propyl side chain are supposed to occur only in
minute amounts in land plants. High amounts (145 µg/g) of DiMeF(11,3) were found by
Hannemann et al. (1989) in the algae Chlorophyta spec.
The amounts of DiMeF(9,5) and DiMeF(11,5) found in green tea were slightly higher
than those published by Guth and Grosch (1993a). The amount and composition of the
FFA in green tea probably depend on provenience and variety. Processing of the green
tea and further treatment could also have an influence on both amount and composition
of the FFA. Depending on the kind and care of processing, degradation and
transformation of the FFA can be more or less advanced. In this context, Guth and
Grosch (1993a) observed a 10-fold amount of pentyl DiMeF in fresh green tea leaves
compared to green tea powder.
The origin of the olefinic derivatives of the DiMeF(11,5) found in most of the
investigated samples is not fully understood. According to Ishii et al. (1988a), olefinic
FFA are artefacts formed during sample preparation. DiMeF(9, 5:1) and DiMeF(9:1, 5)
were found to be generated during the analytical process (GC analysis with split-splitless
injection) from a diketoene formed by autoxidation of the FFA. The olefinic FFA found
in the samples could have been formed from diketoenes already present in the material
as well. However, Boselli et al. (2000) did not agree with the artefact-assumption for
DiMeF(11, 5:1) and DiMeF(11:1, 5) but claimed these olefinic FFA to be of natural
origin in the investigated oil samples.
Results and Discussion
4.2
53
Isolation of furan fatty acid photooxidative degradation products by using micro simultaneous
distillation solvent extraction
In order to study the aroma active furan fatty acid photooxidative degradation products,
it was first necessary to choose a suitable method for the isolation of the volatile
compounds from the heterogenous plant material. As has been described by Parliment
(1997), sample preparation for the analysis of food aroma is complicated by a number of
factors. The generally low concentration level, the complexity, the variation of volatility
and the instability of the aroma compounds as well as the complex food matrices could
create problems during isolation procedures. Various isolation techniques have been
developed ( see e.g. in Schreier (1984), Maarse and Grosch (1996)). However, the
sample preparation remains the most critical step in the entire analytical process of the
investigation of volatile substances (Schreier, 1984) and has to be carefully adapted to
the corresponding problem.
A modified microversion (Godefroot et al., 1981) of the simultaneous distillation
solvent extraction (SDE) apparatus described by Likens and Nickerson (1964) was used
for the isolation of the aroma compounds pentanal, 2,3-butanedione, 2,3-octanedione,
MND, bovolide and dihydrobovolide (subsequently named as analytes). In combination
with the applied OC injection mode, the micro-SDE allowed the direct GC analysis of
the extract without any concentration step.
Since steam distillation can cause thermal reactions (Garneo, 1977), artefacts can arise
from chemical degradation or reactions between the individual volatiles during the
isolation procedure (Schreier, 1984). To ensure that the analytes were not formed during
the micro-SDE, a solution of pentyl DiMeF was subjected to this distillation procedure
(see chapter 3.4.1). GC-MS analysis of this isolate showed that no aroma compounds
were formed during the distillation. Distillation of a mixture of the analytes by microSDE did not show an alteration of the analytes either. Herewith no artefact formation
was caused using the micro-SDE procedure.
Recovery studies with the analytes were performed to evaluate the efficiency of the
sample preparation using micro-SDE. The recoveries (RV) were determined both from
54
Results and Discussion
an aqueous mixture and from a green tea matrix. In Tab. 16 the RV of the analytes and
of the ethyl decanoate (ISTD 1) and the ethyl valerate (ISTD 2) from the two different
media are shown. The RV are reported as percent of the initial peak area ratio of the
analytes to the internal standard methyl undecanoate (ISTD 3).
Table 16: Recoveries (RV) and relative standard deviations (RSD) of the aroma active
volatile furan fatty acid degradation products and of two ethyl esters
extracted by micro-SDE from an aqueous model mixture and from a green
tea matrix
aqueous mixture
green tea matrix
analyte
RV [%]1
RSD [%]
RV [%]2
RSD [%]
pentanal
94.5
4.8
47.3
8.0
2,3-butanedione
83.6
3.2
33.6
5.3
2,3-octanedione
94.5
2.4
76.6
6.4
MND
101.0
4.3
89.2
3.8
bovolide
92.3
2.4
74.4
8.4
dihydrobovolide
76.5
7.6
35.3
12.0
ethyl decanoate (ISTD 1)
91.0
3.6
81.1
6.1
ethyl valerate (ISTD 2)
101.6
2.5
92.0
4.9
1. mean value (n = 5)
2. mean value (n = 6)
The RV of the compounds extracted from the aqueous model mixture reflect the optimal
conditions for the micro-SDE method. The reproducibility of the procedure is
satisfactory with a relative standard deviation of less than 5 % (with one exception).
Most of the analytes were extracted at a high percentage of > 90 %. The low RV of 2,3-
Results and Discussion
55
butanedione (83.6 %) was expected because of the relatively high volatility of this
compound compared to the other analytes. In contrast to 2,3-butanedione, the observed
RV of dihydrobovolide (76.5 %) is probably due to its low volatility in steam.
Experiments with a reduced distillation time of 50 % revealed an incomplete extraction
of both bovolides. However, a considerable extension of the used distillation time
(60 min) would increase the risk of unacceptable losses of the other compounds.
The two ethyl esters (ISTD 1, ISTD 2) were used in subsequent analyses as internal
standards and as reference substances in a controlling system to evaluate the micro-SDE
process. The RV of both substances (> 90.0 %) was satisfactory, and the reproducibility
was very good. ISTD 2 was directly added to the organic solvent. The RV of 101.6 %
indicates an optimal, loss-free recycling of the organic solvent. In addition to an
incomplete extraction from the water phase, a part of the lower RV could only be due to
slight losses of the analytes in the circular flow of the water phase.
The RV of the aroma compounds isolated from a slurry with green tea were much lower
compared to the ones obtained in the model experiment with the aqueous mixture. The
relative standard deviations (RSD) varied between 3.8 % (MND) and 12.0 %
(dihydrobovolide), indicating reproducible losses during the micro-SDE procedure. The
RV of both ethyl esters, ISTD 1 added to the slurry and ISTD 2 added to the organic
solvent, made clear that some losses occurred in both circular flows. The RV of ISTD 1
and ISTD 2 could be taken as indicators for losses of the aroma compounds from these
phases as well. The low RV of pentanal (47.3 %), 2,3-butanedione (33.6 %) and
dihydrobovolide (35.3 %), however, can not be explained by losses due to the microSDE procedure alone. It is very likely that the matrix of the green tea influences the
distillation of the analytes to a different extent.
Despite the low RV of some aroma compounds from the green tea sample the used
micro-SDE was estimated to be suitable because of its reproducibility. However, one
must be aware that the yield of extraction of certain compounds can vary within large
ranges depending on the food sample.
The GC analysis of the compounds extracted by micro-SDE allowed a semi quantitative
evaluation. The results were expressed as peak area ratio to an internal standard. Ethyl
56
Results and Discussion
decanoate (ISTD 1) was shown to be suitable for both sample preparation and GC
analysis. However, it must be taken into account that by using only one ISTD the selfcompensating effect of gain or losses during the extraction procedure can not be the
same for all analytes because of their different chemical and physical properties. In order
to ensure reproducible conditions for sample preparation (and with it constant starting
conditions for the semi quantitative analysis), it was necessary to have the ISTD 1 in a
constant concentration in the extract. To verify this requirement, a controlling system
was developed using two additional standards (ISTD 2 and ISTD 3). Ethyl valerate
(ISTD 2) was added to the organic phase prior to the isolation procedure, and methyl
undecanoate (ISTD 3) was added to the extract after the isolation procedure. In Tab. 17
the mean values (MV) and the RSD of the peak area ratios (RMS) of ISTD 1 and ISTD
2 to ISTD 3 are shown for different series of measurements, consisting of 6 or 9 green
tea extracts from the light exposure experiments (see chapter 4.4).
Table 17: Determination of the reproducibility of the micro-SDE procedure using
green tea samples. Peak area ratios (RMS) for ethyl decanoate and ethyl
valerate to the internal standard methyl undecanoate and corresponding
relative standard deviations
RMS (ISTD 1/ISTD 3)
RMS (ISTD 2/ISTD 3)
series of measurements
MV1
RSD2 [%]
MV
RSD [%]
n3 = 9
0.624
4.8
0.336
5.0
n=6
0.612
7.6
0.320
6.7
n=6
0.627
2.8
0.343
6.3
n=9
0.694
4.0
0.384
6.0
n=6
0.719
3.1
0.391
4.8
n=6
0.591
5.6
0.359
6.7
1. mean value
2. reproducibility expressed as relative standard deviation (RSD)
3. number of analyses
Results and Discussion
57
The calculated RMS values for ISTD 1/ISTD 3 and ISTD 2/ISTD 3 showed good RSD
from 2.8 % to 7.6 % and from 4.8 % to 6.7 %, respectively. These RMS values were used
as indicators for the concentrations of ISTD 1 and ISTD 2 in the extracts. The low RSD
for all sets of experiments made clear that the concentrations of ISTD 1 and ISTD 2 in
the extract were constant, and consequently the micro-SDE process was reproducible.
Additionally, with these two ratios, circulation related problems of the organic and/or
aqueous solution during the micro-SDE procedure could be revealed. Depending on the
losses, the yield of one or both ISTD was affected. In such cases it had to be assumed
that the yield of the aroma compounds was also unacceptably affected and the extraction
had to be repeated.
The use of three ISTD turned out to considerably enhance the quality assurance for the
sample preparation by micro-SDE. Provided that the starting material was
homogeneous, information regarding the reproducibility without making replicate
analyses could be obtained.
4.3
Oxidative stability of furan fatty acid photooxidative
degradation products
Little is known about the oxidative stability of aroma active FFA photooxidative
degradation products. Oechslin (1997) showed that bovolide and dihydrobovolide are
stable under photooxidative conditions. In the present study the photooxidative stability
of the two diones MND and 2,3-octanedione was investigated. Degradation of these two
aroma compounds could lead to other aroma compounds that significantly contribute to
the flavour of food. Therefore the main attention was paid to the light induced formation
of aroma compounds in the oxidative degradation model experiments with MND and
2,3-octanedione.
4.3.1
3-Methyl-2,4-nonanedione
The experiments were carried out in hexane and in methanol as described in chapter 3.6.
The main products obtained in the two solvents under the different experimental
conditions are listed in Tab. 18 and 19.
986
1121
1321
1449
1583
1643
1727
1741
1861
1935
1996
2009
2270
1
2
3
4
5
6
7
8
9
10
11
12
13
0.4
2.4
unknown4
unknown4
-
5.1
46.3
11.4
1.0
0.5
0.7
5.1
0.2
2.0
photooxidation
0.7
r.s
r.s
r.s
MS, IR
r.s
r.s
r.s
r.s
r.s.
identification1
unknown4
BHT
hexanoic acid
MND
HMND
ISTD 1
unknown4
acetic acid
2,3-octanedione
ISTD 2
2,3-butanedione
compound
n.d.
n.d.
n.d.
-
n.d.
102.7
tr.
1.0
4.1
n.d.
0.1
2.8
0.5
1.3
-
7.6
47.5
16.3
1.0
0.3
1.1
7.2
0.2
4.6
n.d.2
0.2
unsensitised
oxidation
dark
oxidation
n.d.
n.d.
n.d.
-
tr.
43.5
n.d.
1.0
5.6
n.d.
tr.3
0.2
n.d.
photochemical
reaction
1. Identification based on the following criteria: r.s.: RI and mass spectra consistent with reference substances; MS: identification based on mass spectral characteristics; IR: identification based on infrared spectral characteristics.
2. not detected
3. traces
4. Mass spectral characteristics see Tab. 20
RI
peak
no.
internal standard ethyl decanoate (ISTD 1)
Table 18: Main products derived from MND under different reaction conditions in hexane, expressed as peak area ratio to the
58
Results and Discussion
986
1068
1129
1189
1323
1443
1577
1638
1722
1740
1859
1935
1947
1
2
3
4
5
6
7
8
9
10
11
12
13
unknown2
BHT
hexanoic acid
MND
HMND
ISTD 1
unknown2
acetic acid
2,3-octanedione
methyl hexanoate
ISTD 2
unknown2
2,3-butanedione
compound
r.s
r.s
r.s
MS, IR
r.s
r.s
r.s
MS
r.s
r.s.
identification1
2.0
-
2.7
35
17.1
1
1.9
0.5
4.8
1.3
0.2
0.2
2.6
photooxidation
n.d.
-
n.d.
63.4
0.6
1
0.2
n.d.3
0.7
0.3
0.2
0.1
0.3
dark oxidation
1. Identification based on the following criteria: r.s.: RI and mass spectra consistent with reference substances; MS: identification based on mass spectral characteristics; IR: identification based on infrared spectral characteristics.
2. mass spectral characteristics see Tab. 20
3. not detected
RI
peak no.
internal standard ethyl decanoate (ISTD 1)
Table 19: Main products derived from MND under different reaction conditions in methanol, expressed as peak area ratio to the
Results and Discussion
59
60
Results and Discussion
Only products with an RMS of > 0.1 (based on TIC) were considered. MND showed to
be stable under oxidative conditions in the absence of light (dark oxidation). No
significant decrease of the dione was observed neither in hexane nor in methanol as
compared to the experiments with light exposure. The GC-MS chromatogram of the
products of MND formed in hexane under photooxidative conditions for 24 h is shown
in Fig. 15, and the corresponding chromatogram in methanol as solvent in Fig. 16,
respectively.
8
7
100
80
60
40
20
1
3
4
2
5
0
5.0
6
10.0
15.0
11
12
9 10
20.0
13
25.0
30.0
35.0
time [min]
Figure 15: GC-MS chromatogram of products derived from MND under
photooxidative conditions in hexane (see Tab. 18)
In both cases only few products were formed. The solvent had little influence on the
product composition. Product type and amount differed only slightly. In both solvents
the same five products were found. Among these compounds the four well known aroma
compounds 2,3-butanedione, 2,3-octanedione, acetic acid and hexanoic acid were
identified.
Results and Discussion
61
10
100
9
80
60
40
5
20
13
1
4
8
6
2 3
0
5.0
10.0
11
12
15.0
7
20.0
25.0
30.0
time [min]
Figure 16: GC-MS chromatogram of products derived from MND under
photooxidative conditions in methanol (see Tab. 19)
The main oxidation product of MND was 3-hydroxy-3-methyl-2,4-nonanedione
(HMND). This substance was shown to be aroma active by using GC-O; the odour
description was rubbery, earthy and plastic-like. The compound was identified based on
the mass spectrum (GC-MS) and the vapour phase infrared spectrum (GC-IR). The mass
spectrum of HMND is shown in Fig. 17. The molecular ion was not observed. The fragment ions at m/z 43, 71 and 99 are formed by α-cleavage, whereas the fragment ions at
m/z 88 and 144 are attributed to McLafferty rearrangements.
62
Results and Discussion
100
43
88
144
80
+H
43
99
71
O
O
60
OH
71
40
20
+H
99
88
144
55
59
0
50
100
150
200
m/z
Figure 17: GC-MS spectrum of HMND
The infrared spectra of HMND and of MND are shown in Fig. 18. According to J.H. van
der Maas (2002, personal communication) the two spectra can be interpreted based on
the spectra of similar molecules as follows: The spectra of MND and HMND show both
a C=O stretching band at 1714 cm-1. For comparison the IR spectrum of 3,3-dimethyl2,4-pentadione (neat, Sadtler 56098) shows a doublet with maxima at ~1720 cm-1 and
~1700 cm-1, but in this molecule the carbonyl groups are trans oriented. The splitting in
the spectrum of 3,3-dimethyl-2,4-pentadione is probably due to a field effect (out-ofphase and in-phase coupling). For HMND the OH stretching band of a free (tertiary)
hydroxyl group can be expected at about 3615 cm-1. Evidently, the hydroxyl group
exhibiting at 3478 cm-1 in the spectrum of HMND (Fig. 18 B) is hydrogen bonded to a
carbonyl group. As a consequence its stretching frequency is lowered and the peak is
broadened. The O···H–O angle in a 5-membered ring is ~120°. The frequency of the
C=O stretching band, however, may remain unaltered, since in a 5-membered ring the
C=O···H angle is roughly 90° (hydrogen bond orthogonal to the carbonyl group). The
C=O stretching bands in the spectra of 1-hydroxy-propanone (neat, Aldrich 13,818-5)
and 3-hydroxy-2-butanone (neat, Aldrich 10,897-9) are found at 1724 and 1716 cm-1.
These data clearly show that intramolecular hydrogen bonding does not affect the C=O
Results and Discussion
63
stretching. Therefore MND and HMND exhibit the C=O stretching peak in the same
region.
Absorbance
A
B
Wave number (cm-1)
Figure 18: GC-IR spectra of MND (A) and HMND (B). For interpretation see text.
64
Results and Discussion
In MND, keto-enol tautomerism may occur. The broad band at 1608 cm-1 in the spectrum of MND (Fig. 18 A) could be due to this phenomenon, but no band is present in the
OH stretching region of the spectrum. 3-Methyl-2,4-hexanedione (neat, Sadtler 56100)
shows a carbonyl doublet as 3,3-dimethyl-2,4-pentadione (neat, Sadtler 56098) (same
pattern, ~ 5 cm-1 red-shifted), the broad 1600 cm-1 band and no clear OH stretching band
in the stretching region as well. However, there is some evidence in the spectrum of 3methyl-2,4-hexanedione that points to the presence of hydrogen bonding. Compared to
the spectrum of 3,3-dimethyl-2,4-pentadione (neat, Sadtler 56098), bands are here less
pronounced, and an appreciable background between 1800 and 900 cm-1 is observed.
The presence of some hydrogen bonding, and the absence of an OH stretching band in
the stretching region in the spectrum of MND can be accepted provided that (1) the ketoform is the dominant one and (2) the OH stretching band is very broad. Furthermore the
hydroxyl group has an enolic (more acidic) character. To sum up the interpretation by
van der Maas the spectra shown in Fig. 18 are consistent with the structures of MND and
HMND, respectively.
The formation of a hydroxylated dione as oxidation product of MND is supported from
literature reports as well. Studies by House and Gannon (1958) have shown that the
oxidation of enolisable β-diketones with peracids leads to α-hydroxy-β-diketones.
Furthermore, in the reaction of enolisable β-diketones with singlet oxygen, the
formation of α-hydroxy-β-diketones has been reported by Yoshioka et al. (1998).
A compound with the same mass spectral characteristics as HMND has already been
mentioned by several authors. Horita and Hara (1986) described an unknown
compound in an investigation on aroma concentrates of green tea after exposure to light.
Braggins (1996) reported about an unknown odour compound of rendered sheep fat
which elutes in the same region as HMND under comparable chromatographic
conditions and showed identical mass spectral characteristics. The odour of the
compound was described by Braggins (1996) as plastic-like. The fact that HMND is a
photooxidative and autoxidative degradation product of pentyl DiMeF was first
recognised by Pompizzi (1999) who also reported this compound to be a Baeyer-Villiger
oxidation product of MND. However, the provisional assignment as an ester made by
Pompizzi (1999) was not correct, and has been revised by the present study.
Results and Discussion
65
Some of the products formed under the different experimental conditions have not been
identified yet. The mass spectral characteristics of these unknown compounds are shown
in Tab. 20.
Table 20: Mass spectral characteristics of unknown products derived from MND under
different reaction conditions in hexane (Tab. 18) and methanol (Tab. 19)
RI
solvent
fragment ion (intensity)
1068
methanol
43 (80), 57 (100), 71 (60), 85 (50)
1577
methanol
43 (100), 55 (20), 71 (65), 99 (90)
1583
hexane
43 (75), 71 (20), 85 (100), 100 (60), 113 (20)
1947
methanol
43 (100), 71 (50), 86 (20), 99 (70), 118 (20), 131 (30)
1996
hexane
43 (50), 99 (100), 114 (50), 170 (20)
2009
hexane
43 (70), 71 (30), 88 (100), 99 (25), 130 (20), 144 (25), 186 (20)
2270
hexane
43( 40), 71 (50), 99 (100), 114 (20), 170 (20)
In control experiments it was shown that HMND, 2,3-butanedione, 2,3-octanedione,
acetic and hexanoic acid are only formed under photooxidative conditions. Without light
exposure (Tab. 18 and 19, dark oxidation), these compounds were either absent or
present in minute amounts only. This is also valid for the unknown compounds listed in
Tab. 18 and 19, with one exception. The unknown compound with RI 1583 formed by
oxidation of MND in hexane (Tab. 18, peak no. 5) is present in much higher amounts in
the dark oxidation compared to the photooxidation.
The influence of the sensitiser on product formation (unsensitised oxidation) as well as
the products formed in absence of oxygen (photochemical reaction) were investigated in
hexane as solvent (Tab. 18). No difference between unsensitised oxidation and
photooxidation was observed. This result was surprising because it was assumed that
66
Results and Discussion
MND can not play the role of a sensitiser. Its absorbance maximum is at 287 nm and the
lamps used for the experiments under photooxidative conditions scarcely emit light at
this wavelength. This result is in contrast to the statement by Matsuura and Saito (1976)
who claimed that photooxidation without sensitiser generally gives a more complex
mixture of oxidation products than photooxidation with sensitiser.
In the photochemical experiment (Tab. 18) trace amounts of 2,3-octanedione and
hexanoic acid were detected, the other compounds formed under photooxidative
conditions were not found. Astonishingly, the unknown compound with RI 1583
(Tab. 18, peak no. 5) was formed in similar amounts as in the dark oxidation. This
remains to be explained.
Following Yoshioka et al. (1998) the formation of HMND can be explained through the
ene reaction of the enol form with singlet oxygen to give the α-hydroperoxy-β-diketone.
This substance is subsequently cleaved to the corresponding α-hydroxy-β-diketone. The
formation of 2,3-butanedione, 2,3-octanedione, acetic and hexanoic acid can formally be
explained by the oxidation of the enolic double bonds of the two most favourable enol
forms of MND (Fig. 19). Oxidation of the 2,3-enol leads to acetic acid and 2,3octanedione, whereas oxidation of the 3,4-enol results in hexanoic acid and 2,3butanedione.
Results and Discussion
67
O
O
MND
O
OH
OH O
2
4
3
2,3-enol
3,4-enol
O
O
HO
O
O
OH
O
2,3-octanedione
3
acetic acid
hexanoic acid
O
2,3-butanedione
Figure 19: Proposed oxidation of the 2,3- and 3,4-enol forms of 3-methyl-2,4-nonanedione (MND) to 2,3-octanedione, acetic acid, hexanoic acid and 2,3-butanedione
Based on the results obtained in the present study the formation of 2,3-octanedione can
not only be explained by the pathway starting from pentyl DiMeF suggested by Pompizzi
(1999), but also by a pathway in which MND is formed as an intermediate.
4.3.2
2,3-Octanedione
The experiments were carried out in hexane and methanol as described in chapter 3.6.
The main products obtained with the two solvents are listed in Tab. 21 and 22. Only
products with an RMS (based on TIC) of > 0.1 were considered. The GC-MS chromatogram of the products of 2,3-octanedione formed in hexane under photooxidative
conditions for 24 h is shown in Fig. 20 and the corresponding chromatogram in
methanol as solvent in Fig. 21, respectively.
998
1052
1069
1120
1138
1271
1320
1452
1507
1600
1647
1857
1932
1
2
3
4
5
6
7
8
9
10
11
12
13
BHT
hexanoic acid
ISTD 1
r.s.
r.s.
r.s.
-
16.0
1.0
0.4
unknown3
1.5
17.8
0.4
r.s
r.s.
unknown3
acetic acid
2,3-octanedione
0.5
unknwon3
0.2
4.7
4.7
0.2
photooxidation
0.3
r.s
r.s
r.s
r.s.
identification1
unknown3
ISTD 2
impurity of solvent
impurity of solvent
pentanal
compound
-
n.d.
1.0
n.d.
n.d.
n.d.
48.1
n.d.
n.d.
0.2
n.d.
-
51.0
1.0
2.6
n.d.
4.9
6.8
n.d.
19.9
0.2
9.2
8.1
0.6
n.d.2
n.d.
unsensitised
oxidation
dark
oxidation
1. Identification based on the following criteria: r.s.: RI and mass spectra consistent with reference substances;
2. not detected
3. Mass spectral characteristics see Tab. 23
RI
peak
no.
to the internal standard ethyldecanoate (ISTD 1)
-
31.4
1.0
4.4
n.d.
1.5
41.6
n.d.
1.4
0.2
14.3
11.9
n.d.
photochemical
reaction
Table 21: Main products derived from 2,3-octanedione under different reaction conditions in hexane, expressed as peak area ratio
68
Results and Discussion
993
1124
1163
1320
1441
1643
1854
1931
1
2
3
4
5
6
7
8
BHT
hexanoic acid
ISTD 1
acetic acid
2,3-octanedione
methyl hexanoate
ISTD 2
pentanal
compound
r.s
r.s
r.s
r.s
r.s
MS
r.s
r.s.
identification1
-
2.2
1.0
0.4
68.8
0.7
0.2
0.4
photooxidation
-
n.d.
1.0
n.d.
51.5
0.1
0.2
n.d.2
dark oxidation
1. Identification based on the following criteria: r.s.: RI and mass spectra consistent with reference substances; MS: identification based on mass spectral characteristics;
2. not detected
RI
peak no.
ratio to the internal standard ethyl decanoate (ISTD 1)
Table 22: Main products derived from 2,3-octanedione under different reaction conditions in methanol, expressed as peak area
Results and Discussion
69
70
Results and Discussion
12
100
7
80
60
3
2
13
40
20
1
45
8
6
11
9
10
0
5.0
10.0
20.0
15.0
25.0
30.0
time [min]
Figure 20: GC-MS chromatogram of products derived from 2,3-octanedione under
photooxidative conditions in hexane (see Tab. 21)
4
100
80
60
40
8
20
1
2
5
3
7
6
0
5.0
10.0
15.0
20.0
25.0
30.0
time [min]
Figure 21: GC-MS chromatogram of products derived from 2,3-octanedione under
photooxidative conditions in methanol (see Tab. 22)
Results and Discussion
71
As in the experiments with MND (chapter 4.3.1), only few products were formed and
the solvent had little influence on the product composition. Pentanal, acetic acid and
hexanoic acid were identified both in hexane and in methanol. Additionally in hexane
four unknown products were formed and in methanol one compound which was tentatively identified as methyl hexanoate was present. The mass spectra of the unknown
compounds are listed in Tab. 23. All these products were absent in the control experiment (dark oxidation) with the exception of methyl hexanoate, which was present in
small amounts.
Table 23: Mass spectral characteristics of unknown products derived from 2,3octanedione under different reaction conditions in hexane (Tab. 21)
RI
solvent
fragment ion (intensity)
1138
hexane
43 (20), 71 (100), 115 (60)
1271
hexane
43 (70), 56 (30), 57 (20), 69 (20), 84 (100), 85 (30)
1507
hexane
43 (100), 71 (20), 99 (40)
1600
hexane
43 (100), 58 (30), 71 (20), 86 (50)
The influence of the sensitiser on product formation (unsensitised oxidation) as well as
possible products formed in absence of oxygen (photochemical reaction) were
investigated in hexane as solvent (Tab. 21). As already observed in the experiments with
MND, the unsensitised oxidation gave the same products as the photooxidation. Only the
two unknown products with RI 1271 (Tab. 21, peak no. 6) and with RI 1507 (Tab. 21,
peak no. 9) were not detected. All other products were formed in even higher amounts
in absence of a sensitiser. A sensitiser activity of 2,3-octanedione must be taken into
consideration. The absorption spectrum of 2,3-octanedione exhibits maxima at 270 nm
and 430 nm. According to Rubin (1969), α-diketones absorb UV-light in the range from
270-300 nm and exhibit a second maximum at 330-540 nm due to n,π* transitions.
These diones may function as sensitisers.
72
Results and Discussion
In the photochemical experiment, the same compounds, except pentanal, were detected
as in the unsensitised oxidation. According to Chen and Ho (1998) the mechanism of the
unsensitised photoreaction with oxygen is similar to that of the photoreaction without
oxygen.
The formation of pentanal can formally be explained by the oxidation of the enolic
double bond of 2,3-octanedione. Oxidation of the 3,4-enol leads to pentanal and pyruvic
acid (Fig. 22). However, the presence of pyruvic acid was not verified.
OH
O
3
4
O
2,3-octanedione
O
3,4-enol
OH
O
pentanal
O
O
pyruvic acid
Figure 22: Proposed oxidation of the 3,4-enol form of 2,3-octanedione to pentanal and
pyruvic acid
The main products of the light induced reactions were acetic acid and hexanoic acid.
Carboxylic acids have been described in the literature as products of photoreactions of
α-diketones (Rubin, 1969).
Results and Discussion
4.4
73
Formation of furan fatty acid photooxidative
degradation products in green tea
The formation rate of the aroma compounds pentanal, 2,3-butanedione, 2,3-octanedione,
MND, HMND, bovolide and dihydrobovolide was investigated in green tea as a
representative of dried green plant material. As described in chapter 3.5 green tea was
exposed to light for a time period of 20 and 25 days respectively, under different
photooxidative conditions. The experiments were carried out at two illuminations
(3‘200 lx and 40‘000 lx) in an air or oxygen atmosphere. As a control, the experiments
were performed under the same conditions in the dark. In Fig. 23 the formation of
pentanal and the two bovolides, and in Fig. 24 the formation of the different diones under
the photooxidative conditions are shown in relation to the initial stage of the samples
before light exposure.
All investigated aroma compounds increased during exposure to light, whereas no
changes were observed in the samples stored in the dark. The formation curves of each
compound in the three light exposure experiments were similar. 2,3-Butanedione
showed the slightest relative change and only small differences between the three experiments. The other compounds followed two patterns. On the one hand, pentanal, 2,3octanedione, bovolide and dihydrobovolide increased continuously during at least 15
days of light exposure. The most pronounced increase took place at the beginning of the
experiment. On the other hand, MND and HMND reached a maximum content after two
days of the light exposure, followed by a considerable decrease during the ongoing of
the experiments. On the whole, the major changes occurred during the first two days of
illumination. Light exposure experiments carried out in oxygen atmosphere gave higher
relative changes when compared to the experiments performed in air at the same illumination.
74
Results and Discussion
Figure 23: Formation of pentanal (A), bovolide (B) and dihydrobovolide (C) in green
tea under photooxidative conditions
Results and Discussion
Figure 24: Formation of the diones MND (A), HMND (B), 2,3-octanedione (C) and
2,3-butanedione (D) in green tea under photooxidative conditions
75
76
Results and Discussion
An increase in the illumination from 3’200 lx to 40‘000 lx - all other experimental
conditions were kept constant - did not result in an enhanced formation of FFA degradation products. On the contrary, less aroma compounds were formed. In addition, the
maximum peaks for MND and HMND and the sharp increase for pentanal, bovolide and
dihydrobovolide were already observed after one day of light exposure. Sandmeier and
Ziegleder (1994) observed a similar effect in their investigations on photooxidation of
chicken soup and broccoli soup powder. This unexpected behaviour can be explained
with the type of light induced oxidation. Green tea contains chlorophyll that can act as
sensitiser, thus the conditions for a photosensitised oxidation are given. It must be kept
in mind that for this type of light induced oxidation the illumination does not have an
influence on the product formation, and no induction phase is observed. In the present
investigation both of these criteria were fulfilled. In samples which had been exposed to
light for one day and subsequently stored in the dark for a period of 80 days, no change
in the aroma compounds was measured (data not shown). This result further supports the
evidence for a photosensitised oxidation. In a photo initiated autoxidation, radicals
would have been generated which would have been expected to react in the dark as well.
In photosensitised oxidations the wavelength of the light source can play an important
role. In the case of chlorophylls, emission of light of wavelengths around 400 nm
(yellow-green) and 650 nm (blue-green) can be crucial, since the absorption maxima of
these natural sensitisers are at 430 nm and 662 nm for chlorophyll a and at 453 nm and
642 nm for chlorophyll b, respectively. According to Thron et al. (2001) wavelengths
around 400 nm have a greater impact on photoreactions sensitised by chlorophyll
derivatives than longer wavelengths. To assess the influence of the illumination, two
types of lamps were used (see Fig. 9 in chapter 3.5.2 and Fig. 10 in chapter 3.5.3). The
sodium vapour-high pressure lamp (40‘000 lx) emitted much less light in the range of
the absorption maxima of the chlorophylls at the lower wavelength range compared to
the fluorescent tube (3’200 lx). Thus a less pronounced excitation of the sensitiser in the
green tea could be expected. The formation of the aroma compounds to a lesser extent
in the experiment with the sodium vapour-high pressure lamp could therefore also be
due to the emission spectrum of the lamp.
Results and Discussion
77
As discussed in chapter 4.3.1, 2,3-butanedione, 2,3-octanedione and HMND are products obtained from MND by a photooxidative process. Thus, a decrease of MND during
a longer period of light exposure and a concurrent increase of the other diones was
expected to occur in the light exposure experiments with green tea as well. As shown in
Fig. 24 C, this was the case. The amount of 2,3-octanedione steadily increased during
the experiments under photooxidative conditions. However, the behaviour of MND,
particularly in relation to the formation of HMND, was not as expected. These two βdiketones showed parallel formation curves. No clear cut explanation of this result can
be given. The formation of MND and/or HMND could possibly be suppressed from the
beginning or in the course of the experiment. It could also be explained through a steady
turnover of HMND. Finally MND could constantly be formed from pentyl DiMeF at the
same rate as it was oxidised to HMND. More investigations are needed for a better
understanding of the complex processes of light induced oxidations in food.
The oxidative behaviour of the pentyl DiMeF of green tea (see Tab. 15, chapter 4.1.2) as
precursors of the aroma compounds was investigated by determination of the decrease
of the pentyl DiMeF during a prolonged light exposure at 3’200 lx in air. Fig. 25 shows
the decrease of the two most abundant pentyl DiMeF during the oxidation procedure,
expressed as relative change compared to the sample before light exposure. Both curves
showed nearly the same pattern, indicating the oxidation rates to be similar for the two
pentyl DiMeF. After 2 days of light exposure, approximately 50 % of the pentyl DiMeF
had reacted. As the oxidation process proceeded, the decrease slowed down
considerably. At the end of the experiment after 20 days, approximately 10 % of the two
pentyl DiMeF were still present. This finding is in contrast to the results described by
Guth and Grosch (1991), who observed a rapid oxidation of DiMeF(9,5) and
DiMeF(11,5) in soy bean oil stored in daylight. After 48 h more than 90 % of the two
FFA were degraded. Furthermore, when the oil was exposed to fluorescent light, the two
pentyl diMeF disappeared rapidly and were not detectable anymore after 25 h. Butter
was rapidly oxidised as well. Only 10 % of the initial concentration of FFA was found
after 48 h illumination with fluorescent light (Guth and Grosch, 1992). In model
experiments by Yurawecz et al. (1997) and by Sehat et al. (1998), who performed
thermical oxidations with F(8,6), the half-life time of this FFA was 35 h.
78
Results and Discussion
Figure 25: Decrease of the two main pentyl DiMeF in green tea during light exposure
(3‘200 lx, air)
The differences between the results obtained with green tea and those with other plant
and animal materials as well as pure FFA could be explained by the presence of
substances in green tea which quench the action of the photosensitiser. Furthermore, the
slower oxidation could also be due to a complicated penetration of light into the dried
plant material. A residual amount of pentyl DiMeF after 20 days of light exposure
corresponds well to the formation rates of the two bovolides. The steady increase of
these two aroma compounds (Fig. 23 B and C) can only be explained if sufficient
amounts of the corresponding pentyl DiMeF are available as precursors during the whole
duration of the light exposure experiment. The observed decrease of the pentyl DiMeF
in green tea further supports the statement regarding a steady turnover of MND during
the photooxidative process.
The importance of single aroma compounds for the overall aroma of a food can be
roughly estimated by consideration of their thresholds. Tab. 24 shows the odour
Results and Discussion
79
thresholds in water of the aroma compounds formed by photooxidation of FFA in green
tea.
Table 24: Odour thresholds of aroma active furan fatty acid photooxidative degradation
products
aroma compound
threshold in water,
reference
orthonasal [µg/kg]
pentanal
12
Buttery et al. (1988)
2,3-butanedione
15
Blank et al. (1991)
2,3-octanedione
110
Sigrist et al. (2000)
MND
0.03
Masanetz and Grosch (1998)
HMND
-1
-
bovolide
2.02
Pompizzi (1999)
dihydrobovolide
5002
Pompizzi (1999)
1. no data available
2. determined in an oil phase
The thresholds vary in a broad range from very low values (MND) to high values
(dihydrobovolide). Based on the thresholds, it can be concluded that MND is the most
important of the seven investigated aroma compounds, followed by bovolide, pentanal
and 2,3-butanedione. The high threshold of dihydrobovolide indicates that this substance
is not important for the overall aroma of green tea exposed to light. The OAV (amount
of aroma compound in relation to its threshold) would help to estimate the relevance of
the single aroma compounds and a possible change of the overall aroma during light
exposure. Unfortunately no OAV could be established, because no absolute amounts of
the aroma compounds could be determined. However, since the threshold for MND is
65 times lower as for bovolide and 400 times lower as for pentanal, the other compounds
would have to increase much more during light exposure to overcome the OAV of MND.
80
Results and Discussion
Once MND has been formed in green tea, further influence of light is not expected to
cause relevant changes in the flavour by the investigated aroma compounds.
4.5
Formation of furan fatty acid photooxidative
degradation products in dried green herbs
Dried herbs and vegetables were exposed to light at 4‘500 lx in air for four days to induce
photooxidation as described in chapter 3.5. In Tab. 25 the effect of light on the pentyl
DiMeF present in the investigated herbs and vegetables is shown. The results are
expressed as peak area ratio (RMS) to the internal standard ethyl decanoate for the seven
aroma compounds. The investigated herbs and vegetables have been shown to be
differently susceptible to light exposure. Tarragon, chervil and chive exhibited the most
significant changes after light exposure, whereas onions were hardly affected at all.
Pentanal, 2,3-butanedione, 2,3-octanedione and the two bovolides were already present
before light exposure (except for dihydrobovolide in savory and onion). This result
suggests that a slight oxidative change of the samples occurred already before light
exposure. In most cases the above mentioned aroma compounds increased during light
exposure, thus indicating an oxidation process to take place. The comparatively high
formation of pentanal was obviously not only due to the oxidation of pentyl DiMeF.
Pentanal is known to be a minor oxidation product of some fatty acids as well (e.g.
Frankel, 1998). In basil, savory and onion no MND and HMND could be detected even
after light exposure. In tarragon, dill, chive and leek very small amounts were found,
whereas in chervil, these compounds increased 3-4 times during light exposure. These
results can be explained by the contents in pentyl DiMeF of the investigated samples, as
shown in Tab. 15 (chapter 4.1.2). Basil, savory and onion are very poor in pentyl DiMeF.
Assuming that a part of the formed MND reacted also to HMND, the amounts of these
aroma compounds could be below the detection limit in these samples. Yet, the
significant increase of MND and HMND in chervil after light exposure agrees well with
the comparatively high amounts of DiMeF(9,5) and DiMeF(11,5) present in this herb.
One additional reason for the low amounts of MND and HMND in most of the herbs and
vegetables has been given by Guth and Grosch (1991). In a model experiment, these
authors showed that only 1.3 % of the methyl ester of DiMeF(9,5) was converted into
MND by photooxidation.
1
3
+
tr.
+
–
tr.
14
+
–
6
7
+
–
1
20
+
–
4
2
+
–
1
–
3
+
1. before light exposure
leek
onion
chive
dill
chervil
savory
1
–
9
+4
basil
1
–1
tarragon
2. traces
1
1
tr.
tr.
4
4
1
1
6
5
1
1
2
1
4
2
tr.
tr.
tr.
tr.
6
1
1
tr.
4
1
tr.
tr.
1
tr.
1
tr.2
2,3-octanedione
RI = 1326
3. not detected
pentanal 2,3-butanedione
RI = 986
RI = 997
light
exposure
sample
light exposure
n.d.
n.d.
n.d.
n.d.
2
1
tr.
tr.
3
1
n.d.
n.d.
n.d.
n.d.
2
n.d.
HMND
RI = 1726
4. after 4 d at 4‘500 lx in air
tr.
n.d.
n.d.
n.d.
1
tr.
tr.
n.d.
4
n.d.
n.d.
n.d.
n.d.
n.d.
1
n.d.3
MND
RI = 1742
2
1
tr.
tr.
7
2
4
1
20
4
2
1
4
3
7
1
bovolide
RI = 2164
1
1
n.d.
n.d.
1
tr.
1
1
5
3
n.d.
n.d.
4
4
1
1
dihydrobovolide
RI = 2192
Table 25: Relative concentrations of aroma compounds (expressed as RMS) in different herbs and vegetables before and after
Results and Discussion
81
82
Results and Discussion
In addition to the FFA, other constituents have to be taken into account by interpreting
the different behaviour of the investigated herbs and vegetables. The presence of e.g.
quenchers and sensitisers can influence the photooxidation process in different ways.
Further experiments with edible roots and leaves of plants should be carried out to
investigate the influence of plant constituents on the formation of the aroma compounds
derived from FFA.
83
5
CONCLUSION AND OUTLOOK
In previously performed model experiments DiMeF were identified as precursors of the
aroma compounds pentanal, 2,3-butanedione, 2,3-octanedione, MND, bovolide and dihydrobovolide formed by oxidative degradation. In this work, the behaviour of DiMeF
and the aroma compounds under light exposure in complex food systems was
investigated. Green tea, tarragon, basil, savory, dill, chervil, chive, onion and leek were
taken as representatives for plant material used as foodstuff. The knowledge obtained by
the model experiments could be confirmed and extended.
The sample preparation was the most critical step in the analysis of the aroma
compounds, since the method for the isolation of the volatile substances from the nonvolatile plant material can strongly influence the result. An adapted micro-SDE method
showed to be suitable for the present work. The method turned out to be simple and
reproducible. Acceptable recoveries were obtained and no artefacts were formed.
Additionally, no concentration step of the isolate was necessary for on column GC
analyses. The use of three ISTD gave important information about the reproducibility of
the micro-SDE process and allowed to obtain concise results without performing
replicate analyses.
Beside the analysis of the aroma compounds, the analysis of the FFA in green tea and
the dried green herbs and vegetables was a matter of interest. The GC-MS/MS technique
was applied for the first time in the analysis of FFA. Ion trap GC-MS/MS allowed a fast
and sensitive identification of the different classes of FFA in the methyl ester extract of
plant lipids. To our knowledge, the occurrence of different classes of FFA in tarragon,
basil, savory, chervil, dill, onion and leek was reported here for the first time.
84
Conclusion and Outlook
In contrast to the model experiments, the investigations with the food samples were
complicated by many factors that could not be influenced or remained unknown. The
following parameters were recognised to exert influence on the formation and the
behaviour of the aroma active FFA degradation products:
Composition of the sample
The amount of the aroma compounds was dependent on the amount and the oxidation
rate of the precursor DiMeF present in the samples. Other constituents such as
sensitisers, quenchers and water were considered to significantly contribute to the
formation of the aroma compounds as well, but were not investigated.
Photooxidative conditions
The formation of the aroma compounds was induced by light exposure in all the
investigated samples. All aroma compounds increased during the two first days of light
exposure. Light exposure in oxygen atmosphere resulted in higher amounts of the aroma
compounds than light exposure in air. The amount of the aroma compounds were
affected by the light sources, which differed in the illuminance and the light emission
spectrum.
Stability of the aroma compounds
MND and 2,3-octanedione were shown in model experiments to react to other aroma
compounds under photooxidative conditions. The aroma compounds which derived
from 2,3-octanedione were pentanal, acetic acid and hexanoic acid. The aroma
compounds which derived from MND were HMND, 2,3-butanedione, 2,3-octanedione,
acetic acid and hexanoic acid. HMND was identified for the first time in this work and
was also detected in the food samples.
Other factors which could have influenced the formation and the behaviour of the aroma
active FFA degradation products could not be considered in this study. Further
investigations with model experiments are needed to gain a better understanding of light
induced formation and changes of the investigated aroma compounds.
With the relative amounts of the aroma active FFA degradation products, no final
conclusion could be drawn on their relevance in the food samples. Sensory analyses
should be conducted to investigate the contribution of the aroma compounds to the
flavour or off-flavour of green tea and the investigated herbs and vegetables. The aroma
Conclusion and Outlook
85
compounds investigated should be considered together with all other aroma active
volatiles in the samples. AEDA in combination with the calculation of OAV would help
to determine the most potent aroma compounds in each sample. Finally, the instrumental
analyses, in particular olfactory analyses, should be confirmed by sensory investigations
on the foodstuff itself.
86
87
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CURRICULUM VITAE
1971
Born on 07 April in Flüelen UR
1978 - 1984
Primary school Flüelen UR
1984 - 1991
Gymnasium Altdorf UR, Matura Type B
1992 - 1998
Studies in Food Science and Technology at the Departement of
Agricultural and Food Science, Swiss Federal Institute of
Technology Zurich; Degree in Food Science and Technology (Dipl.
Lm.-Ing. ETH); Laureate of the Willi-Studer award
1998 - 2002
Ph.D. student and research assistant at the Institute of Food Science
and Nutrition, Swiss Federal Institute of Technology Zurich,
Laboratory of Food Chemistry and Food Technology, Prof. Dr. R.
Amadò