Enzymatic modification of palmarosa essential oil: Chemical analysis and olfactory evaluation of acylated products

Article abstract of DOI:10.1002/cbdv.201300241

We have developed an enzymatic protocol to modify the composition of palmarosa essential oil by acylation of its alcohol components by three different acyl donors at various rates. The resulting modified products were characterized by qualitative and quantitative analyses by gas chromatography, and their olfactory properties were evaluated by professional perfumers. We showed that our protocol resulted in two types of modifications of the olfactory properties. The first and most obvious effect observed was the decrease of the alcohol content, with the concomitant increase of the corresponding esters, along with their fruity notes (pear, most notably). The second and less obvious effect was the expression of notes from minor components ((E)-β-ocimene, linalool, β-caryophyllene, and farnesene), originally masked by the sweet-floral-rose odor of geraniol, present in 70% in the palmarosa essential oil used, and emergence of citrus, green, spicy and clove characters in the modified products. This methodology might be considered in the future as a sustainable route to new natural ingredients for the perfumer. Copyright

Full text of DOI:10.1002/cbdv.201300241

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Enzymatic Modification of Palmarosa Essential Oil: Chemical Analysis
and Olfactory Evaluation of Acylated Products
by Jade Ramilijaona, Elsa Raynaud, Charfeddine Bouhlel, Elise Sarrazin, Xavier Fernandez,
and Sylvain Antoniotti*
´
Institut de Chimie de Nice, UMR 7272, Universite de Nice – Sophia Antipolis, CNRS, Parc Valrose,
FR-06108 Nice cedex 2
(e-mail: sylvain.antoniotti@unice.fr)
We have developed an enzymatic protocol to modify the composition of palmarosa essential oil by
acylation of its alcohol components by three different acyl donors at various rates. The resulting modified
products were characterized by qualitative and quantitative analyses by gas chromatography, and their
olfactory properties were evaluated by professional perfumers. We showed that our protocol resulted in
two types of modifications of the olfactory properties. The first and most obvious effect observed was the
decrease of the alcohol content, with the concomitant increase of the corresponding esters, along with
their fruity notes (pear, most notably). The second and less obvious effect was the expression of notes
from minor components ((E)-b-ocimene, linalool, b-caryophyllene, and farnesene), originally masked by
the sweet-floral-rose odor of geraniol, present in 70% in the palmarosa essential oil used, and emergence
of citrus, green, spicy and clove characters in the modified products. This methodology might be
considered in the future as a sustainable route to new natural ingredients for the perfumer.
Introduction. – The use of enzymes in the synthesis of fine chemicals, from
laboratory to industrial scale, is now in many instances a competitive alternative to
conventional synthesis [1–5], with advantages such as the control of the chirality of the
products [6][7], the mild operating conditions, and the overall sustainable aspects of
the processes [8][9]. Such trends are also observed in the field of flavors and
fragrances, with the additional advantage of providing transformations eligible to the
valuable ꢀnaturalꢁ label [10–13]. Existing enzyme-based methodologies typically deal
with single-molecule manipulation [14–16]. On the other hand, essential oils of various
origins, and more generally, odoriferous natural extracts have been used in fragrances
since perfumery was born. As a consequence of their complex composition, their
olfactory impact on perfuming compositions is also complex [17]. Today, modern
perfumery uses both essential oils and synthetic materials, and innovation in fragrance
chemistry is focused on the use of new sources of natural extracts [18], new techniques
of extraction [19–21], and the design of novel synthetic odorants [22]. However, the
status of essential oil alone does not guarantee the access to the perfumerꢁs shelf, and
superior olfactory properties are necessary. As a result, the price range for essential oils
can vary significantly from a few Euros to more than 10,000 per kg, depending on
supply and demand.
In this context, we have been interested in providing new ingredients for the
perfumer by modifying essential oils with enzymes. In particular, we became interested
ꢂ 2013 Verlag Helvetica Chimica Acta AG, Zꢃrich

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in using simple enzymatic transformations (e.g., lipase-based acylation of alcohols) to
modify inexpensive and available essential oils such as palmarosa essential oil, and to
evaluate the subsequent changes of olfactory properties. Two questions, however, had
to be addressed: 1) what will be the global effect of these multiple changes on the odor
of the mixture, which results in complex relationships between odorants in the matrix,
and 2) how interesting will be these modified extracts as new materials for the
perfumers.
Results and Discussion. – To our knowledge, the use of enzymes in essential-oil
research has been limited so far to the pectinase/cellulase-assisted extraction from
bergamot (Citrus bergamia Risso) peel [23], congolese mansa (Solanum americanum
Miller) [24], rose (Rosa sp.) [25–27], olive [28–30], lemon peel [31], lemongrass
(Cymbopogon citratus Stapf.), and lemon eucalyptus (Eucalyptus citriodora Hook)
[32]. We recently reported the use of an enzymatic strategy based on the use of
horseradish peroxidase (HRP) for the removal of eugenol from rose essential oil
without loss of the organoleptic quality and with a good conservation of the chemical
composition [33]. For the first time, an enzyme-based strategy was proposed for
essential-oil treatment in a model study of the detoxification of natural extracts used in
perfumery and cosmetics.
In the present work, we used the enzymatic modification strategy to chemically
modify an essential oil from palmarosa, rich in geraniol and exhibiting, therefore,
strong sweet-floral-rose notes with lemongrass and straw undertones, and improve its
olfactory quality with the aim of providing new materials for the perfumers. Previously,
the lipase-catalyzed acetylation of palmarosa by AcOH to obtain natural geranyl
acetate was reported, but the nature of the enzyme was not disclosed, and neither the
chemical modifications nor the olfactory impact of the modification were investigated
[34].
Lipase-Catalyzed Acylation of Palmarosa Essential Oil. The lipase-catalyzed
acylation of palmarosa essential oil was performed using Candida rugosa lipase and
following a procedure developed in toluene on similar compounds (Scheme 1) [35].
Using vinyl (ethenyl) esters as acyl donors towards palmarosa essential oil, several
products featuring various yields of esterification could be obtained by varying the
amount of acyl donor used combined with the application of different reaction times
(Table 1).
In general, various yields of acylation could be obtained ranging from 20 to 100% by
using 0.25–3 equiv. of acyl donors involved and in 27–168 h reaction time. The
amounts of the isolated modified essential oils were usually good (33–50 mg),
Scheme 1. Example of Acylation of Geraniol by n Equivalent of Acyl Donor (n 0.25–3) catalyzed by
Candida rugosa lipase (CrL)

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Table 1. Acylation of Palmarosa Essential Oil by Vinyl Esters Catalyzed by Candida rugosa Lipase
Entrya)
Acyl donor
Equiv.
Reaction time [h]
Amount of modified
oil obtained [mg]
Acylation yield [%]b)
1
2
3
4
5c)
6
7
8
9
10
V.A.
V.A.
V.A.
V.A.
V.P.
0.25
0.5
1.5
3
0.75
2
0.2
0.45
1.5
3
27
27
121
168
88
88
73
73
73
33
35
37
33
40
50
49
36
48
37
24
38
80
100
77
100
20
40
84
100
V.P.
V.C.
V.C.
V.C.
V.C.
168
a) Conditions: palmarosa essential oil (50 mg) in toluene (5 ml); temp., 408, rotation in an incubator,
b
1
150 rpm. ) Based on the acylation of citronellol performed in parallel and determined by H-NMR of
the crude mixture. c) Performed in duplicates (1.3% relative error).
relatively to the amount of starting material (50 mg) with a little loss during workup
involving evaporation steps.
Gram-Scale Experiment. Selected reactions could be scaled-up by a factor of 10
starting with 0.5 g of palmarosa essential oil without noticeable loss of efficiency. The
results are compiled in Table 2.
Table 2. Examples of Acylation of Palmarosa Essential Oil by Vinyl Esters Catalyzed by Candida rugosa
Lipase at the Gram Scale
Entrya) Acyl donorb) Equiv. Reaction time [h] Amount of modified Acylation yieldc) [%]c)
oil obtained [mg]
1
2
3
V.A.
V.A.
V.C.
1
1
1
24
96
72
410
422
393
>46
100
58
a) Conditions: Palmarosa essential oil (500 mg) in toluene (5 ml); temp., 408; rotation in an incubator,
c
150 rpm. b) See Table 1. ) Based on the acylation of citronellol performed in parallel and determined by
1H-NMR of the crude mixture.
Modified essential oils could thus be obtained on this scale according to a similar
procedure to obtain partially acylated products, at a yield of 46 and 58% with vinyl
acetate and vinyl crotonate (but-2-enoate), respectively (Entries 1 and 3), and a 100%
acetylated product with a longer reaction time (96 h; Entry 2).
Vinyl esters as acyl donors are very efficient partners in this type of reactions.
Indeed, the enol eliminated being equilibrated to the non-nucleophile acetaldehyde,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
total acylation can be achieved. However, in terms of sustainability of the process and
naturality of the products, other acyl donors should be preferred. We have thus
investigated the use of natural AcOH and AcOEt as acyl donors in a model reaction on
citronellol. Following results recently described in the literature by others, we first
focused on the direct use of AcOH as donor [34]. In spite of a reported esterification
yield of 52.5%, obtained after heating an equimolar mixture of geraniol and AcOH
during 34 h at 608, no conversion of citronellol was observed under the same conditions
with Candida rugosa lipase or Candida antarctica lipase B, and the starting material was
recovered unchanged in 90–96% yield. We next turned our attention to the possible
use of AcOEt, satisfactorily involved as acyl donor in the acylation of tyrosol by
Staphylococcus xylosus lipase [36]. A screening of the conditions was performed, and
the results are collected in Table 3.
Table 3. Screening of the Reaction Conditions for the Acylation of Citronellol by AcOEt in the Presence of
Candida rugosa Lipase
Entrya) AcOEt [equiv.] Conversion [%] Enzyme/substrate (w/w [%]) Acylation yieldb) [%]
1
2
3
4
5
6
7
8
9
3
5
3
33
37
42
60
37
59
62
70
100d)
10
10
20
10
20
50
75
100
60
5
5
10
10
15
32
50
66
100
160c)
160c)
160c)
160c)
160c)
160c)
a) Conditions: citronellol (50 mg) was dissolved in 10 ml of anh. toluene and incubated 24 h at 150 rpm at
408 in the presence of n equivalent(s) of AcOEt and 5 mg of lipase. b) Determined by 1H-NMR.
c) AcOEt was used as the solvent. d) Reaction time of 96 h was required.
Under the conditions previously determined with vinyl esters, the use of AcOEt as
acyl donor did not lead to satisfactory acylation yields (Entries 1–3). Even using
AcOEt as solvent, only 10% acylation was observed in 24 h in the presence of 10% (w/
w) lipase (Entry 4). The acylation yield increased linearly as a function of the amount
of enzyme used, as a result of the saturation of the enzyme by the substrate (Entries 4–
8). Optimized conditions were found with 60% lipase and a reaction time of 96 h to
obtain 100% acylation yield (Entry 9). This last result indicated that a biocatalytic
route to ꢀnatural modifiedꢁ palmarosa essential oil could be envisaged.
Olfactory Evaluation. The olfactory evaluation of the modified palmarosa essential-
oil samples was performed by professional perfumers. In addition to the samples
obtained from acylation reactions, untreated essential oil was evaluated, as well as a
negative control sample consisting in the essential oil submitted to the entire procedure
without enzyme. The results are compiled in Table 4 with details for each sample.
Qualitative and Quantitative GC Analyses. The qualitative and quantitative
chemical compositions of the untreated palmarosa essential oil, the negative control

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Table 4. Olfactory Evaluation of the Modified and Unmodified Palmarosa Essential Oils
Entry
Sample
Acylation rate
[%]
Notes
Interest,
originalitya)
1
2
3
4
5
6
7
8
9
Untreated
–
–
24
38
80
100
76
100
20
40
84
Rose-like, lemongrass, straw
Rose-like, citrus, metal
Citrus, metal, straw, lemon
Rose-like, geranium, floral, citrus
Lemon, spicy, eugenol, frutty, pear, fresh
Pear, green, spicy
Pear, fruity, plastic, rose-like
Fruity, marine, pear, floral
Fruity, plastic, burnt
N.A.
1.0
3.3
5
3
3
Neg. controlb)
Acetylated
Acetylated
Acetylated
Acetylated
Propanoylated
Propanoylated
Crotonylated
Crotonylated
Crotonylated
Crotonylated
3
3.3
2.5
1.5
1
10
11
12
Apple, rose-like, weak
Plastic, nutty, straw, floral, fruity
Burnt, plastic, animalic
100
1
a) From 1 (no interest) to 5 (very interesting). N.A.: not applicable. b) Negative control consisting in
palmarosa essential oil submitted to the entire procedure without enzyme.
without enzyme, and each modified sample was analyzed by combined GC/MS and
GC-FID analyses (Table 5).
In Table 5, we have collected the most representative samples with modified
composition. We could identify 50 compounds accounting for 95.8–100% of the total
chromatogram area. The other results are available as Supplementary Information. The
comparison between untreated essential oil and the negative control, consisting in
submitting the essential oil to the whole reaction process in the absence of enzyme,
showed no significant difference. This result was very important to show that the
differences observed in the modified samples are of enzymatic origin, and that the mass
loss by evaporation does not affect the quantitative composition. Besides the expected
ester (acetate, propanoate, and crotonate), longer chain esters were also identified in
the modified essential oils. The formation of these compounds could be explained by
the presence of their vinyl analogs as impurities in the starting vinyl esters (purity 99,
98, and 98% for V.A., V.P., and V.C., resp.).
As a result of the action of Candida rugosa lipase in the presence of acyl donors, the
geraniol contents, as well as other similar alcohols, decreased, and the corresponding
esters were formed, with increased fruity notes. Interestingly, the decrease in geraniol in
the modified samples allowed other compounds, present at low percentages, to express
their own odor, as identified in the sensory analysis (Table 4). It is the case, for example
for (E)-b-ocimene, farnesene, and linalool, with citrus and green notes (appearing in
the acetylated products at 24 and 38%, resp.), b-caryophyllene with spicy-woody-clove
character (see acetylated sample at 80% with eugenol undertone), and obviously
geranyl acetate with the rose-pear-floral notes and farnesyl acetate with green-floral-
rose notes, both heavily felt in the sensory analysis of the acetylated sample at 100%.
Transformations of these impact odorants into each other could not be neglected, since
C10- and C15-derived compounds are related by simple organic transformations that
could occur in the reaction medium (Scheme 2).

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 2. Possible Relationships between Key Compounds Varying in the Series of Modified Essential
Oils Considered
Vinylogous elimination of the AcO of geranyl acetate could indeed form (E)-b-
ocimene, which leads to linalool after Markovnikov-selective hydration. Similarly,
farnesyl acetate could lead to farnesene, and further to b-caryophyllene by cyclization.
It is worth noting that (E,E)-farnesyl pyrophosphate is the biosynthetic precursor of b-
caryophyllene [37].
Conclusions. – In summary, we have developed a lipase-based strategy to gently
modify palmarosa essential oil and allow the expression of different olfactory notes
from the esters formed, but also from odorants originally present but inhibited by the
large quantity and impact of geraniol. Detailed qualitative and quantitative chemical
analyses have been performed and allowed characterization of the slight differences
between all the modified samples, besides the formation of geranyl esters. This type of
modification might be considered in the future to develop new ingredients for fine
perfumery.
This work was supported by the University of Nice – Sophia Antipolis, the Chemical Institute of Nice
´
(Innovative Projects grant), and the CNRS. We are grateful to Jean-Philippe Paris, Pierre Benard, and
´ ´
Frederic Badie (Payan Bertrand S.A., F-Grasse) for the olfactory evaluation, and to Ms. Audrey Bral for
her technical assistance.
Experimental Part
General. Chemicals such as citronellol and vinyl esters, and the org. solvents were purchased from
SigmaꢀAldrich, and used as received, except toluene, which was dried over CaCl2 and distilled at
atmospheric pressure. Enzymes were purchased from SigmaꢀAldrich (Fluka) and stored at ꢀ188.
Palmarosa essential oil from India was a kind gift of Payan Bertrand S.A. (F-Grasse). Anal. TLC: 0.2-mm
precoated plate silica gel 60 F254 (Merck).1H- and 13C-NMR spectra: BRUKER AC 200, chemical shifts
(d) in ppm relative to TMS at 0 ppm (1 H) or CDCl3 (77.16 ppm; 13C); coupling constants, J, in Hz. GC/
MS: Shimadzu QP2010S-MS chromatograph (EI; 70 eV) equipped with a SLB-5ms cap. column
(thickness, 0.10 mm; length, 15 m; inside diameter, 0.10 mm), temp. program: 808, then 188/min to 2008,
and maintained at this temp. for 30 min.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Acylation of Citronellol. In a 20-ml vial, citronellol (0.32 mmol, 50 mg) and the desired acyl donor
(0.96 mmol) were dissolved in toluene (5 ml), and the lyophilized enzyme (Candida rugosa lipase; 5 mg)
was then added. The vial was capped and the mixture was incubated at 408 and shaked at 150 rpm until
completion, (monitoring by TLC). Workup consisted in decantation, withdrawal of the supernatant,
removal of toluene in vacuo, liquid/liquid extraction with Et2O/aq. 5% Na2CO3, collection of the org.
phase, drying (MgSO4), filtration, and solvent removal. Citronellyl acetate, propanoate, and crotonate
were compared with original standards or with literature data [35].
1
Citronellyl Propionate. H-NMR (200 MHz, CDCl3, 208): 5.10–4.90 (m, 1 H); 3.99 (dt, J¼1.8, 6.3,
2 H); 2.14 (q, J¼7.5, 2 H); 2.00–1.70 (m, 2 H); 1.70–1.10 (m, 11 H); 1.01 (t, J¼7.5, 3 H); 0.79 (d, J¼6.3,
3 H). 13C-NMR (75 MHz, CDCl3, 208): 174.9; 131.7; 125.0; 63.2; 37.4; 35.9; 29.9; 28.0; 26.1; 25.8; 19.8;
18.0; 9.5. EI-MS (70 eV; selected ions): 212 (0, Mþ ), 138 (45), 123 (81), 109 (39), 95 (95), 81 (100), 69
(85), 57 (81), 41 (66).
1
3
4
Geranyl Propionate. H-NMR (200 MHz, CDCl3, 208): 5.50–5.30 (tq, J¼7.2, J¼1.2, 1 H); 5.20–
5.00 (m, 1 H); 4.66 (d, J¼7.2, 2 H); 2.39 (q, J¼7.6); 2.20–2.00 (m, 4 H); 1.76 (m, 6 H); 1.67 (s, 3 H); 1.20
(t, J¼7.6, 3 H). 13C-NMR (75 MHz, CDCl3, 208): 174.9; 142.5; 132.2; 124.2; 118.8; 61.7; 39.9; 28.0; 26.7;
26.1; 18.1; 16.8; 9.6. EI-MS (70 eV; selected ions): 210 (1, Mþ ), 136 (18), 121 (27), 93 (36), 80 (16), 69
(100), 57 (57).
Quantitative GC Analysis. GC Analyses were carried out with an Agilent 6890N gas chromatograph,
under the following operation conditions: vector gas, He; injector and detector temps., 2508; injected
volume, 0.2 ml; split ratio 1/100; HP1 column (J&W Scientific), polydimethylsiloxane (50 mꢁ0.20 mm
i.d., film thickness, 0.33 mm; constant flow, 1 ml/min.) and INNOWAX (polyethyleneglycol, 50 mꢁ
0.20 mm i.d.ꢁfilm thickness 0.4 mm; Interchim, F-MontluÅon). Temp. program: 45–2508 at 28/min and
2508 for 60 min. Retention indices (RIs) were determined with C5 to C24 alkane standards as reference.
Relative amounts of individual components are based on peak areas obtained without FID response
factor correction. Three replicates were performed for each sample. The average of these three values
and the standard deviation were determined for each component identified.
GC/MS Analyses. The GC/MS analyses were carried out with an Agilent 6890N chromatograph
coupled to an Agilent 5973 MS detector (Agilent, F-Massy). Samples were analyzed on a fused-silica cap.
column HP-1 (polydimethylsiloxane, 50 mꢁ0.20 mm i.d.ꢁfilm thickness 0.33 mm, Interchim, F-
MontluÅon) and INNOWAX (polyethyleneglycol, 50 mꢁ0.20 mm i.d.ꢁfilm thickness 0.4 mm; Inter-
chim). Carrier gas, He; constant flow, 1 ml/min; injector temp., 2508, split ratio, 1:100; temp. program:
458 to 2508 or 2308, at 28/min, then held isothermal (20 min) at 2508 (apolar column) or 2308 (polar
column), ion source temp., 2308; transfer line temp., 2508 (apolar column) or 2308 (polar column);
ionization energy, 70 eV; electron ionization mass spectra were acquired over the mass range of 35–
400 amu. Identification of the components was based on computer matching against commercial libraries
(Wiley, MassFinder 2.1 Library, NIST98), laboratory MS libraries built up from pure substances, and MS
literature data [38–43], combined with comparison of GC RI values on apolar and polar column.
Enzymatic Acylation of Palmarosa Essential Oil . In a 20-ml vial, palmarosa essential oil (50 mg) and
the desired acyl donor (1 mmol) were dissolved in toluene (5 ml), and the lyophilized enzyme (Candida
rugosa lipase, 5 mg) was then added. The vial was capped, and the mixture was incubated at 408 and
shaked at 150 rpm until completion. Workup consisted in decantation, withdrawal of the supernatant,
toluene removal in vacuo, liquid/liquid extraction with Et2O/aq. 5% Na2CO3, collection of the org. phase,
drying (MgSO4), filtration, and solvent removal. The reaction progress was estimated by analysis of a
parallel reaction performed simultaneously on citronellol and followed by TLC. The final conversion
yield was determined by 1H-NMR of the same sample after workup.
Olfactory Evaluation by Trained Perfumers. Samples of pure lipase-modified palmarosa essential oil
with three different acyl donors (introducing acetyl, propanoyl, and crotonyl groups) at various
conversion yields were submitted on strips to the evaluation of trained perfumers from Payan Bertrand
S.A.

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Received July 19, 2013