Use of Terpenoids as Natural Flavouring Compounds in Food Industry

Abstract

Terpenoids represent the oldest known biomolecules, having been recovered from sediments as old as 2.5 billion years. Among plant secondary metabolites, they are the most abundant and diverse class of natural compounds. The diversity of terpenoids is probably a reflection of their many biological activities in nature, which has made them a widely used resource for traditional and modern human exploitation. They are usually the main constituents of essential oils of most plants offering a wide variety of pleasant scents from flowery to fruity, to woody or balsamic notes. For this reason terpenoids constitute a very important class of compounds for flavour and fragrance industries, in fact, in the US alone, the demand is forecast to grow 3.7 percent per year to $5.3 billion in 2012. The recent patents on production and extraction of terpenoids commonly used as natural flavouring compounds in food industries are reviewed in the present manuscript.

Full text

Recent Patents on Food, Nutrition & Agriculture, 2011, 3, 9-16 9
1876-1429/11 $100.00+.00 © 2011 Bentham Science Publishers Ltd.
Use of Terpenoids as Natural Flavouring Compounds in Food Industry
Lorenzo Caputi and Eugenio Aprea*
IASMA Research and Innovation Centre, Fondazione Edmund Mach, Food Quality and Nutrition Area, Via E. Mach 1,
38010 S. Michele all'Adige, Italy
Received: September 9, 2010; Accepted: October 12, 2010; Revised: October 15, 2010
Abstract: Terpenoids represent the oldest known biomolecules, having been recovered from sediments as old as 2.5
billion years. Among plant secondary metabolites, they are the most abundant and diverse class of natural compounds.
The diversity of terpenoids is probably a reflection of their many biological activities in nature, which has made them a
widely used resource for traditional and modern human exploitation. They are usually the main constituents of essential
oils of most plants offering a wide variety of pleasant scents from flowery to fruity, to woody or balsamic notes. For this
reason terpenoids constitute a very important class of compounds for flavour and fragrance industries, in fact, in the US
alone, the demand is forecast to grow 3.7 percent per year to $5.3 billion in 2012. The recent patents on production and
extraction of terpenoids commonly used as natural flavouring compounds in food industries are reviewed in the present
manuscript.
Keywords: Flavour, flavouring agent, norisoprenoids, terpenes, terpenoids, terpene synthases.
INTRODUCTION
Terpenes represent the oldest known biomolecules,
having been recovered from sediments as old as 2.5 billion
years [1, 2]. They are also the most diverse class of natural
compounds with more than 40000 different structures
reported to date [3]. This enormous ‘chemodiversity’ can be
considered a peculiar characteristic of these metabolites,
which have been isolated from plant, animal and microbial
species [4].
In plants, some terpenes and their oxygenated forms,
called terpenoids, are involved in primary metabolism,
where they play key and highly diverse roles in cellular
function and maintenance. However, a large number of
structurally diverse terpenes have been characterised as
secondary metabolites of plants, such as those known or
assumed to have specialised functions in mediating antago-
nistic and beneficial interactions amongst organisms in their
environments. For instance, terpenes represent a large
component of volatile floral and fruit scents, adaptations that
plants have evolved to attract pollinators and seed dispersers
in order to maximize their fertilization rates and survival.
Hence, the presence or absence of a scent may have a
substantial impact on the yield of agronomical important
crops [5]. In this review, we will refer to terpenoids to
indicate both terpenes and their oxygenated forms.
Despite the diversity of their functions and structures, all
terpenoids derive from the common 5-carbon building
blocks isopentenyl diphosphate (IPP) and its isomer
dimethylallyl diphosphate (DMAPP), also called isoprene
units. In plants, two distinct pathways produce IPP. The
mevalonate (MVA) pathway which is active in the cytosol
*Address correspondence to this author at the IASMA Research and
Innovation Centre, Fondazione Edmund Mach, Food Quality and Nutrition
Area, Via E. Mach 1, 38010 S. Michele all'Adige, Italy;
Tel: ++39 0461 61 51 36; Fax: ++39 0461 61 52 00;
E-mail: eugenio.aprea@iasma.it
and supplies the precursors for the production of sesqui-
terpenoids (C15) and triterpenoids (C30) (see Fig. (1)). The
2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, also
referred to as the deoxyxylulose-5-phosphate (DXP) path-
way which is active in plant plastids, and supplies pre-
cursors for the production of monoterpenoids, diterpenoids
(C20) and carotenoids (C40) (see Fig. (1)).
Terpenes containing more than one isoprene unit are
usually formed in reactions catalysed by prenyl transferases.
These enzymes catalyse the ‘head-to-tail’ addition of IPP
units to allylic diphosphates, typically DMAPP. These reac-
tions produce the C10, C15, and C20 precursors geranyl
diphosphate (GPP), farnesyl diphosphate (FPP) and
geranylgeranyl diphosphate (GGPP), respectively. Reactions
catalysed by enzymes such as squalene synthase generate
larger terpene precursors by combining the polyprenyl
diphosphates. Terpene synthases catalyse the reaction of the
polyprenyl diphosphates to produce the carbon skeletons of
monoterpenes, sesquiterpenes, diterpenes and so on, gene-
rating the main carbon skeletons. When the main backbones
are completed, additional functional groups are subsequently
added by other enzymes to decorate the main structure,
resulting in the tremendous chemodiversity of the final
products. Typical modifications are: hydroxylation, oxida-
tion, reduction, acylation and glycosylation. The reader is
referred to a recent review by Nagegowda for detailed
overviews of terpene biosynthesis in plants [6].
The diversity of terpenoids is probably a reflection of
their many biological activities in nature, which have made
them a widely used resource for traditional and modern
human exploitation [7]. The use of these compounds, in fact,
has permeated human civilisation since the Egyptians,
contributing significantly to the improvement of quality of
life [8]. There have been many applications of plant terpe-
noids in human society. Pharmaceutical, food and cosmetic
industries have exploited them for their potential as
medicines, flavour enhancers and fragrances. To have an

10 Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 Caputi and Aprea
idea of the economic impact of these compounds in our days,
it is worth mentioning that the demand for flavours and
fragrances in the US is forecast to grow 3.7 percent per year
to $5.3 billion in 2012 [9].
In the past decade, the increase in consumption of
fortified food and nutraceutically enriched beverages has
challenged the flavour research as it has provided new
applications for flavour compounds that are able to enhance
the sensory appeal of these products while masking unplea-
sant tastes and aromas of vitamins, minerals, antioxidants
and other added active ingredients. However, the big value
gain of the flavours market seems to be the increasing
demand for ‘natural’ ingredients which are generally more
expensive than their ‘natural-like’ or synthetic counterparts.
The current legislation in the US and EU classifies ‘natural’
flavours as only those that are extracted from natural sources
or obtained through bioprocesses involving precursors
isolated from nature [10, 11]. This classification has led to a
preference for the development of more efficient methods for
the extraction of essential oils from plants and to the
development of new synthetic methods involving biotech-
nology and biotransformation rather than the use of
chemistry. However, for some compounds, such as menthol,
for which the demand greatly exceeds the supply from natu-
ral sources, research on its chemical synthesis is still active.
Another aspect that needs to be pinpointed here is that
currently the majority of the ‘natural’ flavouring agents are
extracted from plants grown through conventional agricul-
tural practices which are strictly dependent on geographical
and seasonal variations, political interference and other
environmental factors. This production system sometimes
does not ensure a continuous product supply and/or a
uniform quality and yield causing significant fluctuations in
the prices of the raw material which inevitably affects the
cost of the products.
In this paper, we will review the sensory properties of
some commercially relevant terpenoids used as flavours
focusing on the recent advances in their extraction or
production methods.
SENSORY PROPERTIES OF COMMERCIALLY
RELEVANT TERPENOIDS
Terpenoids are the major constituents of essential oils,
which are widely used directly as flavouring agents [12-15]
or for further isolation of flavouring substances [16-21]. The
ranges of flavours and odours for these compounds are very
broad and it is not possible to define common descriptors.
Furthermore, several of these compounds exist in different
enantiomeric forms that differ either in odour sensations, or
in odour intensity [22].
In general, monoterpene hydrocarbons such as - and -
pinene, limonene, -3-carene, -phellandrene and myrcene
are found as complex mixtures in most essential oils,
particularly in those extracted from plant leaves, whilst seed
and flower oils contain more specialised monoterpenes and
present fruity or flowery odours. Woody oils contain high
percentages of sesquiterpenes and sesquiterpenols with more
Fig. (1). Schematic overview of terpenoid biosynthesis in plants. In the plastid, isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP), generated through the deoxyxylulose-5-phosphate (DXP) pathway, condense to form terpenoid precursors, including
the monoterpenoid precursor geranyl diphosphate (GPP), and the diterpenoid precursor geranylgeranyl diphosphate (GGPP). Two molecules
of GGPP condense to form carotenoids. In the cytosol, IPP and DMAPP, generated through the mevalonate (MVA) pathway, condense to
form the sesquiterpenoid precursor farnesyl diphosphate (FPP). Two molecules of FPP condense to form the triterpenoids.

Terpenoids as Natural Flavourings in Food Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 11
woody and balsamic notes [23]. Examples of flavouring
substances isolated from essential oils and widely used in
flavour and fragrance industries are menthol from wild mint
(Mentha arvensis) having a strong minty odour; D-carvone
from caraway (Carum carvi) with its spicy and breadlike
odour; D-limonene from citrus species having a fresh orange
peel odour; citral from lemongrass (Cymbopogon citratus)
having a fresh lemon peel odour; 1,8-cineole from
eucalyptus (Eucalyptus globulus) having a camphoraceous-
cool odour. Differently from monoterpenes and sesquiter-
penes that are biosynthetically originated directly from
isoprene units through the MEP and the MVA pathways, the
C13-norisoprenoid compounds such as ionones and damas-
cones are products of the oxidative cleavage of -carotene
and are important sources of flavours for the food industry as
well. These compounds constitute essential aroma notes in
tea, grapes, roses, tobacco, and wine also showing a wide
range of odour attributes. It is worth mentioning here some
of the most common norisoprenoids. -Ionone is present in
many fruits and plants such as black currant, black tea,
blackberries, raspberries, carrots and has an intense violet
odour; -damascenone is mainly present in apricot, rose,
grape, kiwi, raspberries, blackberries and is described as
woody, sweet, fruity, earthy with green floral nuances;
safranal is mainly found in saffron, osmanthus, black tea,
mate, and paprika and has a woody, spicy, phenolic, cam-
phoreous, medicinal odour.
EXTRACTION, PURIFICATION AND CHEMICAL
SYNTHESIS
Many plants contain considerable amounts of terpenoids
hence some parts of the plants, such as seeds, flowers, fruits,
leaves and roots, are used as starting material for the
extraction. Physical processes for isolation of natural
flavouring substances include distillation, solvent extraction
(including the use of dense gases), and chromatography [24].
To isolate low volatile terpenes with polar groups from
plants, fungi and other organisms, the natural material is
dried, chopped or ground and then extracted with inert
solvent at the lowest possible temperature in order to prevent
the formation of artefacts [25]. The extract is separated by
distillation, evaporated to dryness in vacuum or freeze-dried
and then chromatographically fractioned. Enantiomeric
active forms can be further resolved via fractional crystalli-
sation.
(-)-Menthol (see Fig. (2) for structure) or 1-menthol is
one of the most important flavouring chemicals and it is used
extensively in pharmaceuticals, cosmetics, toothpastes,
chewing gum, and other toiletries as well as in cigarettes.
The main source of (-)-menthol is the oil of Mentha arvensis
that is frozen to obtain crystals of menthol which are then
separated by centrifugation. Since the demand for menthol
greatly exceeds the supply from natural sources, consi-
derable effort has been devoted to the production of 1-
menthol by synthetic or semi-synthetic means from other
more readily available raw materials [26-30].
Menthone (see Fig. (2) for structure) exists in two
stereoisomeric forms, menthone and isomenthone, which,
having two asymmetric carbons, occur as pairs of enantio-
mers. Menthone has a typical minty, peppermint-like odour
while isomenthone has a musty, sweet, herbaceous, earthy-
camphoraceous, hay-like odour [31]. Menthone and
isomenthone are constituents of the essential oils of penny-
royal, peppermint, Pelargonium geraniums, and others.
Menthol is added to confectionaries or beverages not only
for its minty odour but also as a cooling and refreshing agent
[32-34]. Menthone can be obtained by distillation of corn
mint oil, by oxidation of menthol [35, 36] or by hydro-
genation of thymol.
As the compound responsible for the flavour of caraway,
dill and spearmint, carvone (see Fig. (2) for structure) has
been used for millennia in food [37]. Carvone is present in
two enantiomeric forms: R-(+)-carvone smells like spearmint
while S-(-)-carvone smells like caraway [38]. In the past, R-
and S- carvones were extracted and isolated by fractional
distillation of caraway oil and spearmint oil, respectively. In
recent years, more efficient extraction methods have been
proposed [39, 40] but nevertheless the synthetic production
is preferred. In the late fifties and during the sixties, several
works were published describing carvone chemical synthesis
[37] but one of the most recent patents is dated 2002 [41].
The preferred starting material for carvone production is
limonene [42].
Although limonene (see Fig. (2) for structure) is mainly
used as a precursor of carvone it is also used as a flavouring
agent in foods, beverages and chewing gum [43]. It is found
in non-alcoholic beverages (31ppm), ice cream and ices
(68ppm), candy (49ppm), baked goods (120ppm), gelatines
and puddings (48-400ppm), and chewing gum (2300ppm)
[43]. Limonene is a chiral molecule, and biological sources
produce D-limonene ((+)-limonene). The main industrial
source of this compound is orange peel, from which it is
extracted using organic solvents [44].
Because of its flavourful and fragrant properties, linalool
(see Fig. (2) for structure) is added to processed food and
beverages [45-47], perfumes, cosmetics and soaps as well as
to household detergents and waxes. The Food and Drug
Administration allows the use of numerous natural and
artificial flavourings in beverages, candy, ice cream and
baked goods containing various amounts of linalool and its
esters. The GRAS list of flavouring ingredients published in
1965 lists linalool and nine of its common esters. Linalool
has a chiral centre at C3 and therefore two stereoisomers are
present: (R)-(-)-linalool and (S)-(+)-linalool. Plants produce
both enantiomeric forms. S-linalool is found, for example, as
a major constituent of the essential oils of coriander
(Coriandrum sativum L.) seed, palmarosa (Cymbopogon
martinii var martinii (Roxb.) Wats), and sweet orange
(Citrus sinensis Osbeck) flowers. (R)-Linalool is present in
Ho oils from Cinnamomun camphora [48], rosewood oil,
lavender (Lavandula officinalis Chaix), laurel (Laurus
nobilis), and sweet basil (Ocimum basilicum, family Lamia-
ceae), among others. Each enantiomer possesses a distinct
scent: S-(+)-linalool is perceived as sweet, floral, petitgrain-
like and the R-form as more woody and lavender-like.
Today, the major portion of linalool on the market is
synthesized either from naturally occurring pinene [49] or
entirely synthetically through a series of complex reactions
[50].

12 Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 Caputi and Aprea
Eucalyptol (see Fig. (2) for structure), or 1,8-cineole, has
a pleasant camphor-like smell, spicy aroma and taste, and is
thus used in flavourings, fragrances, and cosmetics. Cineole
based eucalyptus oil is used as a flavouring at low levels
(0.002%) in various products, including baked goods,
confectionery [51], meat products and beverages. Eucalyptol
with purity from 99.6 to 99.8 percent can be obtained in
large quantities by fractional distillation of eucalyptus oil or
can be synthetically prepared from -terpineole [52].
Nootkatone (see Fig. (2) for structure) is used for
flavouring beverages and is the most important and
expensive aromatic compound of grapefruit. It has been
isolated from grapefruit peel and juice. Nootkatone can be
prepared from oxidation of valencene [53, 54] and recently
its production from ()--pinene has been proposed [55]. It
exits in two enantiomeric forms: (+) nootkatone is the
natural produced form and has a strong grapefruit odour
while the synthetic ()-nootkatone shows a weak woody and
spicy flavour with no grapefruit character [56, 57].
Damascenone (see Fig. (2) for structure) is characterised
by an intense “stewed apple” odour described as woody,
sweet, fruity, earthy with green floral nuances. Its charac-
teristic floral scent contributes to the odour character of
Bulgarian rose oil, tobacco, black tea, raspberry oil and
many other fruits. Biochemically, is a product of the -
carotene degradation but it is industrially synthesised from
pironenes [58]. Most recently a process for conducting an
organic reaction in ionic liquids for the production of -
damascenone and -ionone has been patented [59].

- and

-Ionone (see Fig. (2) for structures) are aroma
compounds found in a variety of essential oils, including
rose oil and are widely used by the flavour industry [60],
hence their importance in foods. -ionone is also a raw
material for the production of retinol. Similarly, to damas-
cenone, the ionones derive from the degradation of carote-
noids [61]. They are the main volatile compounds found in
the headspace of raspberry fruits [62, 63].
The -ionone is sometimes used in conjunction with
anethole, liquorice or menthol to flavour sugar confectionery
Fig. (2). Chemical structures of some terpenoid flavouring compounds.
O
Eucalyptol
HO
Linalool
O
Nootkatone
-Limonene
O
Carvone
HO
Menthol
O
p-Menthone
O
-Ionone -Ionone
O
O
-Damascenone
O
Safranal
O
O
Geranyl isobutyrate
O
O
trans-Geranyl acetate

Terpenoids as Natural Flavourings in Food Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 13
(candy), and both are typically included in raspberry-
flavoured beverages [64]. As for other economically valued
chemicals, methods for their synthetic production have been
patented too [59, 65, 66].
Safranal (see Fig. (2) for structure) is the most abundant
volatile component and the major flavour compound present
in Saffron (Crocus sativus L.) [67] and its natural bio-
chemical origin is from carotenoids degradation. Saffron is
considered to be the most expensive spice in the world and
nowadays its main use is as a foodstuff [67]. Safranal for
aroma or food additive is extracted from saffron [68] or
synthetically prepared [69]. Its odour is described as
"saffron, dried hay like".
Geranyl esters (see Fig. (2) for structures) are essential
oils widely used in food industry [70] and most of them have
a hint of rose. Geranyl acetate is a liquid with a fruity rose
odour reminiscent of pear added in small amounts to fruity
aromas. Geranyl isobutyrate also has a fruity rose odour and
usually is used in combination with the acetate ester.
BIOTECHNOLOGICAL PRODUCTION
In the last few years, metabolic pathway engineering in
both plants and whole-cell systems (plant cell cultures and
microorganisms) and the discovery of new enzymes have
received great attention for the production of commercially
relevant terpenoids. Research in this field has been driven by
the fact that flavouring compounds produced from natural
raw materials by microbial or enzymatic methods can be
labelled as 'natural', thereby satisfying the consumer request
of 'bio' or 'natural' products in the food sector. Furthermore,
the biotechnological approach could also ensure a conti-
nuous product supply through a well standardized process
with high productivity.
This strategy has been largely supported by the recent
boost in genome sequencing projects which have provided
vast resources for gene discovery. In fact, to date, many
patents have been filed reporting new genes involved in
terpenoids biosynthesis, especially terpene synthases. The
interest in this class of enzymes arises from the fact that they
are responsible for the wealth of terpene carbon skeletons
found in nature. In addition, some terpene synthases are able
to generate multiple products starting from the same
substrate, hence producing a complex aroma. For instance, a
sesquiterpene synthase has been recently discovered that
produces a mixture of sesquiterpenes composed of -santa-
lene, epi--santalene, cis--bergamotene, trans--bergamo-
tene, and endo--bergamotene [71]. Also, bifunctional enzy-
mes capable of efficient formation of both monoterpenes and
sesquiterpenes depending on substrate availability have been
recently discovered [72]. A list of terpene synthases patented
in the last decade and their activities is given in Table 1 [71,
73-84]. For an up-to-date list of monoterpene and sesqui-
terpene synthases reported in literature we refer the reader to
a recent review [85]. However, the engineering of these
genes in plants and whole-cell systems (both plant cells and
microorganisms) has revealed that in order to increase the
metabolic flux through the pathway and to obtain significant
yields of products further pathway engineering is required.
In particular, it is necessary to remove possible metabolic
bottle-necks and to provide additional flux of substrates by
acting on earlier biosynthetic steps. Recently, it has been
reported that increasing the level of IPP available through
engineering of hydroxymethylglutaryl-CoA reductase
(HMGR), the rate-controlling enzyme of the MVA pathway,
led to a significant increase of terpenes production in both
microorganisms [86] and plants [87]. Further, it has been
shown that the overexpression of small subunit of geranyl
diphosphate synthase, the enzyme responsible for the
formation of the precursor of monoterpenes, led to signifi-
cant increase of such compounds [88]. Examples of host
organisms engineered with a complete heterologous pathway
that enhances the terpenoids production have also been
reported [89].
C13-Norisoprenoids are compounds naturally present in
plants at very low concentrations deriving from the oxidative
cleavage of carotenoids by the action of carotenoid cleavage
dioxygenase (CCD) enzymes. A recent invention [90]
describes the engineering of microorganisms with plant
CCDs to produce - and -ionone, theaspirone, -damas-
cenone, -damascone.
Research has been also carried out on the identification
and exploitation of the enzymes responsible for further
modifications of the terpene backbones, since they are
responsible for a greater structural diversity. In particular,
attention has been devoted to cytochrome P450 enzymes
which can hydroxylate terpenes [91, 92], reductases [93] and
to glycosyltransferases, enzymes responsible for the forma-
tion of monoterpenoid [94] and sesquiterpenoid glycocon-
jugates [95].
The metabolic engineering of both microorganisms and
plants for the biotechnological production of relevant terpe-
noids has seen, in the last decade, enormous advancements
in terms of yields of the products making this technology
more attractive for industrial processes. It is worth
mentioning that in some cases the advancements have been
so fast that, for instance, within about a year the yield of the
sesquiterpenoid farnesol produced in the bacterium E. coli
has been increased from 389 g/L [96] to 135.5 mg/L [97].
Metabolic engineering has also allowed the production in
good yields of other relevant prenyl alcohols, such as
nerolidol [98], geranylgeraniol [98], geraniol [99], and
cubebol [100, 101] in yeasts. Several attempts in advancing
plant systems as efficient terpenoids production platforms
have also been made. Although the first results were not very
encouraging, recent work report significant improvements of
product yields, suggesting that this approach also represents
a valid alternative route for terpenoids production. For
instance, the expression of a heterologous limonene synthase
in tobacco only led to the production of 40 ng/g of product in
the cytoplasm and 143 ng/g in the plastids [102]. However,
the work of Wu et al. [103] showed that by applying a more
sophisticated engineering strategy, in which carbon flux is
redirected by targeting the cell compartment not normally
used for that class of isoprenoids, it is possible to signi-
ficantly increase the amount of product formed. In fact, the
co-expression of a limonene synthase with a geranyl
diphosphate synthase targeted to the cytosol or to the plastid,
led to the production of 374 ng/g and 513 ng/g, respectively.
The same work also reported a 25000-fold increase in
sesquiterpene amorpha-4,11-diene synthesis using the same
strategy.

14 Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 Caputi and Aprea
The results published in this area are very encouraging
and have already attracted the attention of companies
specialising in flavours and fragrances production. In fact,
several patents reported in this paper concerning the
biotechnological production of terpenoids have been filed by
industries which are also directly participating in several
ongoing studies on the topic.
CURRENT AND FUTURE DEVELOPMENTS
The patents reviewed in this paper reveal that in order to
face the increasing demand of flavouring agents new
extraction and production methods are being developed.
Typically, current approaches involve extractions from plant
sources and chemical synthesis. In the first case, the supply
of raw material, usually obtained through conventional
agricultural practices, can be limited and not sufficient to
support the increasing demand of ‘natural’ flavourings.
Synthetic chemistry, on the other hand, can provide new
efficient routes to the production of flavourings but these
products can only be labelled as ‘natural-like’ and their price
will be, in most of the cases, lower than their ‘natural’ coun-
terparts. Whilst the research on these methods continues,
there is also an increasing interest in the development of new
alternative approaches based on biotechnologies. In fact, the
recent legislation that regulates the classification of
flavouring products has led to a preference for synthetic
methods involving biotransformation rather than the use of
chemistry. We believe that the future development in this
area will probably rely on the combination of cutting-edge
research in gene discovery and metabolic engineering but a
big contribution could be provided by the discovery of new
flavouring agents through the accurate analysis of the
‘metabolome’ of plant species, including those currently
used for extraction. Furthermore, it should be mentioned that
the engineering of plants to increase terpenoids production
could also be exploited to increase the nutritional value of
food crops or to enhance the plant fitness itself.
ACKNOWLEDGEMENTS
The authors thank Dr. Urska Vrhovsek (IASMA
Research and Innovation Centre, Food Quality and Nutrition
Area) for reviewing the manuscript. Lorenzo Caputi is
supported by the MetaQuality project, funded by the
autonomous Province of Trento (Italy).
CONFLICT OF INTEREST
The authors have no conflict of interest.
REFERENCES
[1] Summons RE, Jahnke LL, Hope JM, Logan GA. 2-
Methylhopanoids as biomarkers for cyanobacterial oxygenic
photosynthesis. Nature 1999; 400: 554-7.
[2] Brocks JJ, Logan GA, Buick R, Summons RE. Archean molecular
fossils and the early rise of eukaryotes. Sci 1999; 285:1033-6.
[3] Roberts SC. Production and engineering of terpenoids in plant cell
culture. Nat Chem Biol 2007; 3: 387-95.
[4] Rohdich F, Bacher A, Eisenreich W. Isoprenoid biosynthetic
pathways as anti-infective drug targets. Biochem Soc Trans 2005;
33: 785-91.
[5] Dudareva N, Pichersky E. Biochemical and molecular genetic
aspects of floral scents. Plant Physiol 2000; 122: 627-33.
[6] Nagegowda DA. Plant volatile terpenoid metabolism: Biosynthetic
genes, transcriptional regulation and subcellular compartmentation.
FEBS Lett 2010; 584: 2965-73.
[7] Bohlmann J, Keeling CI. Terpenoid biomaterials. Plant J 2008; 54:
656-69.
[8] Zwenger S, Basu C. Plant terpenoids: Applications and future
potentials. Biotechnol Mol Biol Rev 2008; 3: 1-7.
Table 1. Recently Patented Mono- and Sesquiterpene Synthases
Patent no. Enzyme type Product(s) Ref:
US20100138954 Sesquiterpene synthase
-Santalene, epi--santalene, cis--bergamotene, trans--bergamotene, endo--
bergamotene
[71]
US20100209969 Monoterpene synthase Geraniol [73]
US20090205060 Sesquiterpene synthase Patchoulol, -curcumene, germacrane-type sesquiterpenes [74]
US20100144893 Mono-, sesquiterpene synthase Linalool, nerolidol [75]
US20100216186 Sesquiterpene synthase Valencene [76]
US7273735 Sesquiterpene synthase Valencene, bicyclogermacrene, cubebol, -cadinene [77]
US20020038001 Sesquiterpene synthase Bisabolene, -selinene, -humulene [78]
US20030166204 Sesquiterpene synthase (E)--farnesene [79]
US6342380 Sesquiterpene synthase Germacrene [80]
US20030175861 Monoterpene synthases (-)-Camphene, (-)--phellandrene, terpinolene, (-)-limonene, (-)-pinene [81]
US20090313718 Sesquiterpene synthases Caryophyllene [82]
US20060137032 Sesquiterpene synthases -Farnesene [83]
US20060162008 Sesquiterpene synthase Germacrene-D, -cadinene, -cadinene, -muurolene, -elemene, -elemene,
elemol, germacrene-B
[84]

Terpenoids as Natural Flavourings in Food Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 15
[9] Freedonia Industry Research. Report: US Flavors & Fragrances
Market. Available at: http://www.reportlinker.com/p0100133/US-
Flavors-Fragrances-Market.html (Accessed on: September 8,
2010).
[10] US Code of Federal Regulations 21, 101.22a.3. Food and Drug
Administration, Washington, D.C. 1985.
[11] The Council of the European Communities Council Directive
88/388/EEC of 22 June 1988.
[12] Reiss, I., Erfurt, H., Ott, F., Kindel, G., Gabriel, B. Process for the
preparation of a flavoring concentrate, and a flavoring concentrate.
US20090155446 (2009).
[13] Closson, A., Monteleone, M.G. Organoleptic compounds.
US20090156456 (2009).
[14] Dewis, M.L., Huber, M.E., Agyemang, D.O., Conklin, G.
Compositions of oxo-terpene derivatives and process for preparing
the same. US20090180973 (2009).
[15] Franklin, L., Cloud, E., Knapp, L., Ostroff, G., Harman, G.
Terpene-containing compositions and methods of making and
using them. US20100136102 (2010).
[16] Takagaki, R., Yamada, Y. Bisabolane type sesquiterpene
composition and its production. JP9111282 (1997).
[17] Asakawa, Y. Aroma composition. US4720354 (1988).
[18] Stroganov, I.A. Method of production of special vodka.
RU2171281 (2001).
[19] Guntert, M., Kindel, G., Koppe, V., Lambrecht, S., Surburg, H.
Mint and/or fruit flavor compositions. US6322838 (2001).
[20] Dewis, M.L., Huber, M.E., Agyemang, D.O., Conklin, G.
Compositions of oxo-terpene derivatives and process for preparing
the same. US7534460 (2006).
[21] Bowen, H.R. Selective purification of mono-terpenes for removal
of oxygen containing species. US20060100470 (2006).
[22] Brenna E, Fuganti C, Serra S. Enantioselective perception of chiral
odorants. Tetrahedron-Asymmetry 2003; 14: 1-42.
[23] Bensouilah J, Buck P. Aromadermatology: Aromatherapy in the
treatment and care of common skin conditions. 1st ed. Radcliffe
Publishing Ltd: Oxford 2006.
[24] Ziegler E, Ziegler H. Flavourings: production, composition,
applications, regulations. 1st ed. Wiley-VCH: Weinheim, Germany
1998.
[25] Breitmaier E. Terpenes: flavors, fragrances, pharmaca,
pheromones. 1st ed. Wiley-VCH Verlag GmbH & Co. KGaA:
Weinheim, Germany 2006.
[26] Chaplin, J.A., Gardiner, N.S., Mitra, R.K., Parkinson, C.J.,
Portwig, M., Dickson, M.D., Brady, D., Marais, S.F., Reddy, S.
Process for preparing (-)-menthol and similar compounds.
WO0236795 (2002).
[27] Gatfield, I.L., Hilmer, J.M., Bornscheuer, U., Schmidt, R., Vorlova,
S. Method for preparing D- or L-menthol. EP1223223 (2006).
[28] Bergner, E.J., Ebel, K., Johann, T., Lober, O. Method for the
production of menthol. US7709688 (2010).
[29] Sayo, N., Matsumoto, T. Method for producing 1-menthol.
US6342644 (2002).
[30] Gralla, G., Heydrich, G., Bergner, E.J., Ebel, K. Process for
preparation of menthol by hydrogenation of isopulegol.
US20100191021 (2010).
[31] Enberger R, Hopp R. In: Berger RG, Nitz S, Schrier P, Eds. Topics
in flavor research. Marzling, Germany: H Eichorn 1988: 201-18.
[32] Shimizu, T., Shigeta, Y., Kunieda, S. Food products containing a
fruit component. US20070092623 (2007).
[33] Wolf, F.R., Mcgrew, G.N., Hook, J.S., Richey, L.C., Patel, M.M.,
Yatka, R.J., Witkewitz, D.L., Greenberg, M.J., Tyrpin, H.T.,
Nelson, K.T. Chewing gum containing physiological cooling
agents and method of making. US20080199565 (2008).
[34] Witkewitz, D.L., McGrew, G. N. Combinations of cooling agents
for use in confections. US7767243 (2010).
[35] Odinokov KIV, Botsman LP, Emel'yanova GA. The synthesis of
menthone by ozonization of menthol. Russian Chem B+ 1998; 47:
2021-2.
[36] Haut SA. A convenient preparation of pure menthol and menthone
isomers. J Agric Food Chem 1985; 33: 278-80.
[37] De Carvalho CCCR, Da Fonseca MMR. Carvone: Why and how
should one bother to produce this terpene. Food Chem 2006; 95:
413-22.
[38] Leitereg TJ, Guadagni DG, Harris J, Mon TR, Teranishi R.
Chemical and sensory data supporting the difference between the
odors of the enantiomeric carvones. J Agric Food Chem 1971; 19:
785-7.
[39] Chemat S, Aït-Amar H, Lagha A, Esveld DC. Microwave-assisted
extraction kinetics of terpenes from caraway seeds. Chem Eng
Process 2005; 44: 1320-6.
[40] Chemat S, Lagha A, Aït-Amar H, Bartels PV, Chemat F.
Comparison of classical and ultrasound-assisted extraction of
carvone and limonene from caraway seeds. Flav Frag J 2004;
19:188-95.
[41] Davey, P.N., Newman, C.P., Thiam, W.A., Tse, C.-L. Preparation
of carvon. US6500989 (2002).
[42] Linder SM, Greenspan FP. Reactions of limonene monoxide - the
synthesis of carvone. J Org Chem 1957; 22: 949-51.
[43] NTP. National Toxicology Program technical report on the
toxicology and carcinogenesis studies of d-limonene (CAS
No.5989-27-5) in F344/N rats and B6C3F1 mice (Gavage Studies),
NTP TR 347, NIH Publication No. 90-2802, 1990.
[44] Herve, A., Durand, G., Durand, F. Plant extract obtained by an
extraction method by means of solvents of plant origin.
US20090306386 (2009).
[45] Vilas, P.S., Venkatraj, V.N. Process for preparing a tea beverage.
US20090304895 (2009).
[46] Colliver, S.P., Cooper, M.A., Griffiths, A., Peilow, A.D., Sharp,
D.G. Method for the manufacturing of a green tea product.
US20070160737 (2007).
[47] Shimizu, T., Shigeta, Y., Kunieda, S. Food products containing a
fruit component. US20070092623 (2007).
[48] Xiangyun, L. Method for distilling and extracting natural linalool
from Cinnamomum camphora leaves. CN101619013 (2010).
[49] Semikolenov VA, Ilyna II, Simakova IL. Linalool synthesis from
-pinene: Kinetic peculiarities of catalytic steps. Appl Catal A-Gen
2001; 211: 91-107.
[50] Ohwa M, Kogure T, Eliel EL. An asymmetric synthesis of
enantiomerically pure (S)-(+)-linalool (3,7-dimethyl-1,6-octadien-
3-ol) and a formal synthesis of (R)-(-)-linalool. J Org Chem 1986;
51: 2599-601.
[51] Hayade, T., Fujimoto, T. Chewing gum using spice containing
menthol and cineole. JP9327266 (1997).
[52] Leão Lana EJ, da Silva Rocha KA, Kozhevnikov IV, Gusevskaya
EV. Synthesis of 1,8-cineole and 1,4-cineole by isomerization of a-
terpineol catalyzed by heteropoly acid. J Mol Catal A-Chem 2006;
259: 99-102.
[53] Furusawa M, Hashimoto T, Noma Y, Asakawa Y. Highly efficient
production of nootkatone, the grapefruit aroma from valencene, by
biotransformation. Chem Pharm Bull 2005; 53: 1513-4.
[54] Huang, R., Christenson, P.A., Labuda, I.M. Process for the
preparation of nootkatone by laccase catalysis. US6200786 (2001).
[55] Sauer, A.M., Crowe, W.E., Laine, R. A., Gregg, H. Efficient and
economic asymmetric synthesis of nootkatone, tetrahydronoot-
katone, their precursors and derivatives. US7112700 (2006).
[56] Haring HG, Rijkens F, Boelens H, Van Der Gen A. Olfactory
studies on enantiomeric eremophilane sesquiterpenoids. J Agric
Food Chem 1972; 20: 1018-21.
[57] Boelens MH, Boelens H, van Gemert LJ. Sensory properties of
optical isomers. Perfumer Flavorist 1993; 18: 1-15.
[58] Boulin B, Tarana M, Arreguy-San Miguela B, Delmond B. New
preparation methods for -damascone, -damascone, and -
damascenone using pyronenes. Synth Commun 2007; 37: 2579-91.
[59] Bourgeois, D.M., Miracle, G.S., Porter, P.J., Boros, E., Seddon,
K.R., Gunaratne, H.Q.N. Process for conducting an organic
reaction in ionic liquids. WO2010080505 (2010).
[60] Pickenhagen, W., Smith, A.Y. Flavoring with mixtures of ionones.
US4247572 (1981).
[61] Winterhalter P, Rouseff RL. In: Winterhalter P, Rouseff RL, Eds.
Carotenoid-derived aroma compounds: An introduction.
Washington, DC: ACS Symposium Series 802: 1-17.
[62] Aprea E, Carlin S, Giongo L, Grisenti M, Gasperi F.
Characterization of 14 raspberry cultivars by solid-phase
microextraction and relationship with gray mold susceptibility. J
Agric Food Chem 2010; 58: 1100-5.
[63] van Gemert LJ. Compilations of odour threshold values in air and
water. TNO Nutrition and Food Research Institute, Boelens Aroma
Chemical Information Service (BACIS), Huizen, The Netherlands
1999.
[64] Burton, G., Daroszewski, J. Food supplements and uses thereof.
US20090306222 (2009).

16 Recent Patents on Food, Nutrition & Agriculture, 2011, Vol. 3, No. 1 Caputi and Aprea
[65] Yamamoto, T., Yagi, K., Ishida, K. Method for producing optically
active alpha-ionone. US20090216039 (2009).
[66] Dobler, W., Bahr, N., Breuer, K., Kindler, A. Continuous process
for producing pseudoionones and ionones. US7141698 (2006).
[67] Maggi L, Carmona M, del Campo CP, Kanakis CD, Anastasaki E,
Tarantilis PA, et al. Worldwide market screening of saffron volatile
composition. J Sci Food Agric 2009; 89: 1950-4.
[68] Agarwal, S.G., Thappa, R.K., Agnihotri, V.K., Suri, O.P., Qazi,
G.N. Method for the extraction of saffron pigments and flavor
concentrate. US7070823 (2006).
[69] James, H.B. Methods for preparing aldehydes by self-aldol
condensation. US20090270658 (2009).
[70] Johnson, W.E., Gorman, C.R. Process for making terpene esters.
US7355066 (2008).
[71] Sallaud, C., Rontein, D., Tissier, A. Genes encoding Z,Z-farnesyl
diphosphate synthase and a sesquiterpene synthase with multiple
products and uses thereof. US20100138954 (2010).
[72] Nagegowda DA, Gutensohn M, Wilkerson C, Dudareva N. Two
nearly identical terpene synthases catalyze the formation of
nerolidol and linalool in snapdragon flowers. Plant J 2008; 55: 224-
39.
[73] Pichersky, E., Iijima, Y., Lewinsohn, E., Gang, D.R., Simon, J.
Geraniol synthase, methods of production and uses thereof.
US20100209969 (2010).
[74] Schalk, M., Deguerry, F. Sesquiterpene synthase from patchouli.
US20090205060 (2009).
[75] Aharoni, A., Jongsma, M.A., Verhoeven, H.A., Bouwmeester, H.J.
Isoprenoid synthases. US20100144893 (2010).
[76] Chappell, J., Greenhagen, B. Novel sesquiterpene synthase gene
and protein. US20100216186 (2010).
[77] Schalk, M., Clark, A. Sesquiterpene synthases and methods of use.
US7273735 (2007).
[78] Croteau, R.B., Bohlmann, J., Crock, J.E., Steele, C.L.
Sesquiterpene synthases from grand fir (Abies grandis), and
methods of use. US20020038001 (2002).
[79] Croteau, R.B., Crock, J.E. Isolation and bacterial expression of a
sesquiterpene synthase cDNA clone from peppermint (Mentha x
piperita, L.) that produces the aphid alarm pheromone (E)-beta-
farnesene. US20030166204 (2003).
[80] Colby, S.M., Crock, J.E., Lemaux, P.G., Croteau, R.B. Germacrene
C synthase gene of Lycopersicon esculentum. US6342380 (2002).
[81] Steele, C.L., Bohlmann, J., Croteau, R.B., Phillips, M.A. Mono-
terpene synthases from grand fir (Abies grandis). US20030175861
(2003).
[82] Degenhardt, J., Kollner, T., Gershenzon, J. Polynucleotides
encoding caryophyllene synthase and uses thereof.
US20090313718 (2009).
[83] Green S. A., Friel E. N., Beuning L. L., Macrae E. A. Plant alpha
farnesene synthase and polynucleotides encoding same.
US20060137032 (2006).
[84] Green S., Friel E., Beuning L., Macrae E. Enzymes and
polynucleotides encoding the same. US20060162008 (2006).
[85] Degenhardt J, Köllner TG, Gershenzon J. Monoterpene and
sesquiterpene synthases and the origin of terpene skeletal diversity
in plants. Phytochemistry 2009; 70: 1621-37.
[86] Keasling, J.D., Newman, J.D., Pitera, D.J. Method for enhancing
production of isoprenoid compounds. US20090004724 (2009).
[87] Chappell, J., Wu, S., Schalk, M., Clark, A. Transformed plants
accumulating mono-and/or sesquiterpenes. US20080201796
(2008).
[88] Orlova I, Nagegowda DA, Gutensohn M, Kish CM, Maeda M,
Varbanova M, et al. The small subunit of snapdragon geranyl
diphosphate synthase modifies the chain length specificity of
phylogenetically distant geranylgeranyl diphosphate synthase in
planta. Plant Cell 2009; 21: 4002-17.
[89] Clark, A., Maury, J., Asadollahi, M.A., Møller, K., Nielsen, J.,
Schalk, M. Method for producing terpenes and MEP-transformed
microorganisms thereof. US20090155874 (2009).
[90] Beekwilder, M.J., Sibbesen, O., Mikkelsen, J.D., Meer, I.M.V.D.,
Hall, R.D., Qvist, I. Enzymes. US20070259397 (2007).
[91] Aharoni, A., Jongsma, M.A., Bouwmeester, H. Terpene
hydroxylation. US20090031455 (2009).
[92] Ro, D., Newman, K., Paradise, E.M., Keasling, J.D., Ouellet, M.,
Eachus, R., Ho, K., Ham, T. Polynucleotides encoding isoprenoid
modifying enzymes and methods of use thereof. US20100218283
(2010).
[93] Croteau, R.B., Davis, E.M., Ringer, K.L. Isolated menthone
reductase and nucleic acid molecules encoding same.
US20090300801 (2009).
[94] Caputi, L., Lim, E.K., Bowles, D.J. Monoterpenoid modifying
enzymes. US20100143975 (2010).
[95] Caputi, L., Lim, E.K., Bowles, D.J. Sesquiterpenoid modifying
enzymes. US20100132073 (2010).
[96] Ohto C, Muramatsu M, Obata S, Sakuradani E, Shimizu S. Prenyl
alcohol production by expression of exogenous isopentenyl
diphosphate isomerase and farnesyl diphosphate synthase genes in
Escherichia coli. Biosci Biotechnol Biochem 2009; 73: 186-8.
[97] Wang C, Yoon SH, Shah AA, Chung YR, Kim JY, Choi ES, et al.
Farnesol production from Escherichia coli by harnessing the
exogenous mevalonate pathway. Biotechnol Bioeng 2010; 107:
421-9.
[98] Otho C, Muramatsu M, Obata S, Sakuradani E, Shimizu S.
Overexpression of the gene encoding HMG-CoA reductase in
Saccharomyces cerevisiae for production of prenyl alcohols. Appl
Microbiol Biotechnol 2009; 82: 837-45.
[99] Oswald M, Fisher M, Dirninger N, Karst F. Monoterpenoid
biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res 2007;
7: 413-21.
[100] Asadollahi MA, Maury J, Patil KR, Schalk M, Clark A. Enhancing
sesquiterpene production in Saccharomyces cerevisiae through in
silico driven metabolic engineering. Metab Eng 2009; 11: 328-34.
[101] Asadollahi MA, Maury J, Schalk M, Clark A, Nielsen J.
Enhancement of farnesyl diphosphate pool as direct precursor of
sesquiterpenes through metabolic engineering of the mevalonate
pathway in Saccharomyces cerevisiae. Biotechnol Bioeng 2010;
106: 86-96.
[102] Ohara K, Ujihara T, Hendo T, Sato F, Yazaki K. Limonene
production in tobacco with Perilla limonene synthase cDNA. J Exp
Bot 2003; 54: 2635-42.
[103] Wu S, Schalk M, Clark A, Miles BM, Coates R, Chappell J.
Redirection of cytosolic or plastidic isoprenoid precursors elevates
terpene production in plants. Nat Biotechnol 2006; 24: 1441-47.