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© 2000 Institute of Food Technologists

Food Chemistry and T

oxicology

JFS:

Food Chemistry and Toxicology

Headspace Volatiles and Physical Characteristics
of Vacuum-microwave, Air, and Freeze-dried
Oregano (Lippia berlandieri Schauer)

A.N. Y

OUSIF

, T.D. D

URANCE

, C.H. S

CAMAN

,

AND

B. G

IRARD

ABSTRACT: Mexican oregano (Lippia berlandieri Schauer) was dried using a freeze drier, conventional hot air drier,
and a recently developed vacuum-microwave drier. The effect of the drying method on the relative content of major
flavor volatiles, rehydration rate, color, and structural integrity of the herb was evaluated. Dynamic headspace
analysis of volatiles present in fresh or dried oregano revealed that

b-myrcene, a-terpinene, g-terpinene, p-cymene,

and thymol were the major volatile compounds of the plant. The level of thymol in vacuum-microwave-dried
oregano was found to be comparable to that of fresh or freeze-dried samples (1.3 times the thymol concentration of
air-dried). Air-dried samples of oregano were darker, were less green, and exhibited lower rehydration rates than
those prepared by vacuum-microwave or freeze-drying. Structures of vacuum-microwave and freeze-dried oregano
appeared to be quite similar as observed on electron micrographs.

Key Words: oregano, volatiles, vacuum-microwave, drying

Introduction

A

SURVEY

OF

THE

LITERATURE

REVEALED

that numerous plants in various plant

families are known colloquially as orega-
no, oreganum, or some similar name. This
herb is a perennial plant which is charac-
terized by its pungent spicy odor. The
herb is marketed in the fresh or dried
states. The air-dried plant and its extract
are used in flavoring vegetables, meats,
sauces, and other food products.

Generally, air drying of aromatic herbs,

including oregano, is an effective method
of preservation that inhibits the growth of
microorganisms and delays the onset of
some biochemical reactions in the final
product. Hot air drying, however, can
cause thermal damage and can severely
modify the physical and chemical charac-
teristics of the marketed product. Al-
though freeze drying can be used to avoid
damage caused by heat, producing a
product with superior physical and chemi-
cal qualities, it is considered a costly and
time consuming process.

Vacuum-microwave drying offers an al-

ternative way to improve the quality of de-
hydrated products. The low temperature
and fast mass transfer conferred by vacu-
um (Huxsoll and Morgan 1968), combined
with rapid energy transfer of microwave
heating, generates very rapid, low-tem-
perature drying. The absence of air during
drying may inhibit oxidation. Therefore,
physical properties such as structure, col-
or, and sensory qualities of products can
be better preserved. To date, vacuum-mi-
crowave drying has been successfully
used in the dehydration of animal materi-

als such as shrimp (Lin and others 1998b),
and krill (Durance 1997), as well as plant
materials such as potato chips (Durance
and Liu 1997), carrots (Lin and others
1998a), cranberries, ( Yongsawatdiguul
and Gunasekaran 1996a,b), and sweet ba-
sil (Yousif and others 1999). Regardless of
their nature, vacuum-microwave-dried
foods showed better retention of key con-
stituents and better sensory properties
than air-dried equivalents.

This study was undertaken to investi-

gate the potential use of vacuum-micro-
wave methods for drying oregano, and to
compare the effect of various drying
methods on the physical and chemical
qualities of this aromatic herb.

Materials and Methods

Plant source

Mexican oregano, Lippia berlandieri was

purchased from a local wholesale market
in Surrey, B.C, but it was originally pro-
duced in and imported by air from the
United States. The herb was generally
available with the stems and leaves at-
tached. In all experiments, the whole
plant was used, excluding the roots. The
initial moisture of the plant material was
82.4% on a wet weight basis.

Drying

A sample (600 g) of fresh oregano was

placed in the drying drum (constructed
from high-density polyethylene) of a 4 kW
maximum power microwave vacuum
chamber (26 in by 20 in) SS 304 stainless
steel, EnWave Corp., Vancouver, B.C.,

Can.). The drum was rotated at a rate of 11
rotations per min. After a vacuum of 27 in
of Hg was achieved, the magnetron was
powered at 3.2 kW for 12 min, followed by
1 kW for 6 min and then 0.5 kW for 5 min.
Microwave power was measured by the
IMPI 2-L test (Buffler 1993). Under these
conditions, the temperature of the materi-
al was 45 °C as measured at the end of the
drying period using an Infrared Ther-
mometer (Model 39650-04, Cole Parmer
Instruments Co., Chicago, Ill., U.S.A.). Am-
bient airflow rate through the chamber
was 3 L/min. The final moisture content
and water activity of the plant material
were 13.2% and 0.55, respectively.

Fresh oregano from the same batch

was air-dried using a commercial dryer as
follows: 1 kg of fresh basil leaves was load-
ed on a Vers-a-belt dryer (Wal-Dor Indus-
tries Ltd., New Hamburg, Ont.). The dryer
temperature was set at 48 °C with an air
flow rate of 2.3 m

3

/s and relative humidity

of 25%. After 11.5 h in the dryer, moisture
content and water activity of the plant ma-
terial were 13.2% and 0.53, respectively.

A 3rd sample of fresh oregano was

freeze-dried under vacuum (1.6 mm Hg).
Chamber and condenser temperatures
were 20 °C and -55 °C, respectively. The
dried material had a moisture content and
water activity of 9% and 0.54%, respective-
ly.

Water activity (a

w

) and moisture

content determinations

Water activity (a

w

) of all dried samples

was determined using the Aqualab (model
CX-2, Decagon Devices, Inc., Pullman,

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Wash., U.S.A.). Moisture content of dry
samples was determined (in triplicate) us-
ing a laboratory oven at 103 °C. Samples
were dried to a constant weight.

Headspace volatile compounds
analysis

Volatile compounds of oregano were ex-

tracted and collected by a dynamic head-
space technique, separated on a Varian
3700 gas chromatograph (Varian Associ-
ates, Inc., Palo Alto, Calif., U.S.A.), and
identified by gas chromatography-mass
spectrometry (GC-MS).

Six samples of fresh or dried oregano

were weighed in clean Zip-lock bags so
that the content of each bag delivered a fi-
nal concentration of 0.6% (w/v, based on
moisture content studies) when suspend-
ed in the preheated (60 °C) distilled water
contained in aone l purge and trap appa-
ratus ( Wheaton, Millville, N.J., U.S.A.).
The temperature of the apparatus was
held at 60 °C throughout the experiment
by circulating water from a water bath.
Fresh samples were blended (using a
household blender) in 100 mL of preheat-
ed (60 °C) distilled water until completely
homogenized (30 sec) and then placed in
a purge and trap apparatus that con-
tained 400 mL of preheated water (60 °C).
Dried samples of the herb, were crushed
while inside the sealed plastic bag, and
the flakes were immediately added to 500
mL of 60 °C water in the purge and trap
vessels. An internal standard, tetradecane
(Aldrich Chemicals, Milwaukee, Wis.,
U.S.A.) dissolved (1:100) in diethyl ether
(BDH Chemicals, Toronto, Ont.) was add-
ed (500 uL) to each of the vessels that were
attached to a horizontal shaking platform.
The headspace of the shaken herb-con-
taining vessels was purged with purified
N

2

(Linde Specialty Gas, Vancouver, B.C.)

at 50 mL/min for 2 h and passed through
an adsorbent trap containing Tenax GC
(60-80 mesh, Alltech Co., Deerfield, Ill.,
U.S.A.). About 100 mg of Tenax GC was
packed into each glass tube (18 cm, 6 mm
o.d., 4 mm i.d.), which was secured at both
ends with glass wool deactivated with SY-
LONÔ-CT (Supelco Inc., Toronto, Ont.).
The Tenax GC was conditioned prior to
first use as recommended by the manu-
facturer. Subsequent use of the traps was
preceded by stripping the adsorbent with
diethyl ether and drying at 60 °C with N

2

flowing at 30 mL/min for 30 min. Diethyl
ether (2 mL) was used to elute the volatile
compounds from the Tenax GC, and the
extract was concentrated to approximately
300 uL by directing a gentle stream of N

2

onto the surface. A sample (1 uL) of the
concentrated extract was injected into the
GC that was equipped with a flame ioniza-
tion detector (FID) and a polyethylene

glycol (PEG) capillary Supelcowax-10 col-
umn (30 m, 0.25 mm i.d., 0.25 um film
thickness, Supelco Inc., Toronto, Ont.).
The column temperature was held at 35 °C
for 5 min, programmed at 4 °C per min to
200 °C and held at 200 °C for 5 min. The in-
jector port and detector were set at 220 °C
and 250 °C, respectively. The flow rates for
helium (carrier gas) and hydrogen gas
were set at 30 mL/min and for air at 300
mL/min. Splitless injection was employed.
Data was collected and processed with the
JCL 6000 Chromatography Data System
for PC ( Jones Chromatography, Lake-
wood, Colo., U.S.A.). The relative amount
of major volatile compounds was deter-
mined by dividing the area of a compound
by peak area of the internal standard and
multiplying by 100.

For identification of the volatile com-

pounds, separation was performed using
a longer (60 m) Supelcowax-10 fused silica
capillary column that was housed in a
Hewlett-Packard 5890-5970 GC-MSD sys-
tem (Hewlett-Packard, Avondale, Pa.,
U.S.A.). Oven temperature was initially
held at 35 °C, then increased by 3 °C/min
to 200 °C with a final hold time of 10 min.
The carrier gas was helium and the column
head pressure was maintained at 30 psi.
The MSD operating conditions were scan
range 25 to 200 amu, threshold 400, sam-
ple rate 2.7 scan/s, and EM voltage 1800.
Mass spectral identification was obtained
with a HP G1034C MS Chem Station con-
taining a HP G1035A Wiley (138.1) PBM li-
brary.

Color

All dried samples and a commercially

available air-dried sample were analyzed
colorimetrically. Five grams of each treat-
ment samples were ground (in triplicate)
in a household coffee grinder for 10 s to
produce a powder of a uniform color. The
samples were then transferred to a 10 cm
Petri dish, and subsequently read by a
Hunter LabScan II Spectrocolorimeter
(HunterLab, Reston, Va., U.S.A.). The in-
strument, equipped with a D

65

illuminant

and 2 ° observer optical position, was stan-
dardized using a black plate and a stan-
dard white plate (No. LS-13685, X = 79.8, Y
= 84.67, Z = 91.23). The results were ex-
pressed as HunterLab L (whiteness/dark-
ness), a (red/green), and b (yellow/blue)
values.

Rehydration

The rehydration potential of dried

oregano leaves was evaluated by immers-
ing pre-weighed samples, held by plastic
netting in small glass containers in water
at (1) 30 °C and (2) 100 °C. The samples (in
triplicate for each time interval and treat-
ment temperature) were drained under

vacuum (in a Buchner funnel, for 30 sec.)
and re-weighed at 0, 10, 20, 30, 60, 90, and
120 min for samples rehydrated at 30 °C;
and at 0, 0.5, 1.0, 4.0, 6.0, 8.0, and 10.0 min
for samples rehydrated at 100 °C. The
weight of water absorbed (g) divided by
the dry sample weight (g) was expressed
as the rehydration ratio. The slope of the
rehydration ratio against rehydration time
was defined as the rehydration rate.

Scanning Electron Microscopy
(SEM)

Dried whole leaves of similar size and

moisture content were chosen at random
from the various drying methods for scan-
ning electron microscopic examination.
The leaves were fragmented, under a dis-
section microscope, into smaller pieces.
Leaf fragments were then attached to SEM
stubs and subsequently coated with gold
(about 25 nm) using the Nanotech SEM-
PREP II Sputter Gold Coater, and finally
stored under desiccation until examined
by the scanning electron microscope (Ste-
reoscan 250, Cambridge Instruments Ltd.,
Cambridge, U.K.). Polaroid pictures were
taken and processed as specified by the
manufacturer.

Statistical analysis

Statistical analysis software (InStat for

MacIntosh, version 2.01) was used to eval-
uate the significance of the difference be-
tween the various treatment groups. Stu-
dent t-test was used to compare the mean
values of the various treatments. Mean
values were considered significantly dif-
ferent when p < 0.05.

Results and Discussion

A

ROMA

LOSS

IS

ONE

OF

THE

IMPORTANT

changes that occur during drying of

herbs. Aroma is an important quality fac-
tor that influences consumer acceptability
of herbs. In oregano, flavor is mainly de-
termined by one or both of its two charac-
ter-impact compounds, thymol and/or
carvacrol. The literature shows a wide vari-
ation in the essential oil composition and
concentration of oregano. The concentra-
tion of thymol and carvacrol shows an in-
teresting pattern depending on the region
where oregano is produced. Oregano used
in this research was classified as belonging
to the family Verbenaceae, a cultivar indig-
enous to Mexico and the southern United
States. Mexican oregano is characterized
by its high content of thymol, present as
the main constituent at 40% to 60% of total
volatiles, and carvacrol at 5 to 25%
(Lawrence 1984). Monoterpene hydrocar-
bons such as

b-myrcene, a-terpinene, g-

terpinene, and p-cymene are also present
in Mexican oregano oils at concentrations
between 20% and 40%, with g-terpinene

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Effect of Drying on Characteristics of Oregano . . .

and p-cymene being the most prevalent.

The aroma of a food product is deter-

mined by detection of volatile compounds
which escape from the food matrix and are
present in the headspace above the food
material. Volatiles can be released to the
headspace at varying concentrations de-
pending upon their solubility in the ma-
trix, absolute amount, and volatility.
Therefore, trapping analysis of the head-
space volatiles, as used in this study, can
be more closely related to the sensory aro-
ma profile of the product than solvent ex-
traction of total volatiles.

More than twenty four volatiles collect-

ed by the headspace method were detect-

ed in oregano by capillary GC. Com-
pounds that were identified are listed in
Table 1. A typical chromatogram of head-
space flavor volatiles of fresh oregano is
shown in Fig. 1. In this study, the peak ar-
eas (relative abundance) of most of the
identified compounds, including carvac-
rol, were small. Therefore for further dis-
cussion only the quantitatively major
compounds found in the sample analyzed
were considered, including the main aro-
ma volatiles, for example,

b-myrcene, a-

terpinene,

g-terpinene, p-cymene, and

thymol.

Figure 2 shows the effect of the drying

method on the abundance of the five vol-

atile compounds. The level of monoterpe-
nes,

b-myrcene, and a-terpinene, in the

air or vacuum-microwave-dried samples
was not significantly different from levels
found in the fresh samples. The signifi-
cant increase in concentration of

b-

myrcene upon freeze drying is notewor-
thy. A combination of enhanced loss of
this monoterpene during air or vacuum-
microwave drying as compared to freeze
drying, and /or greater hydrolysis of non-
volatile conjugates of the freeze-dried
samples during the volatiles’ extraction in
the purge and trap apparatus may be re-
sponsible for this elevation in concentra-
tion. This phenomenon is known to occur
among various volatile compounds of aro-
matic herbs (Braja and others 1989; Stahl-
Biskup 1987) although the mechanisms
responsible for these observations is not
understood. The amount of

g-terpinene

showed a significant decrease from origi-
nal levels present in the fresh samples
when the plant was air- or vacuum-micro-
wave-dried, but not freeze-dried.
P-Cymene concentration in fresh oregano
was significantly reduced upon drying by
all of the three methods. Thymol, a key
character-impact compound, was also af-
fected by air-drying. Statistical analysis
showed a significant reduction in thymol
concentration in air-dried samples, while
its concentration remained unaltered in
vacuum-microwave or freeze-dried sam-
ples. Although formal organoleptic testing
of the samples dried by the various meth-
ods was not performed, it was evident that

Table 1—Headspace volatile compounds from
oregano, L. berlandieri extracted and col-
lected by headspace procedure, analyzed and
identified by GC/MS

Peak no.

a

Compound

1

a

-Pinene

2

b

-Thujene

3

p-Xylene

4

m-Xylene

5

o-Xylene

6

3-Heptanone

7

b

-Myrecene

8

a

-Terpinene

9

Limonene

10

b

-Phellandrene

11

g

-Terpinene

12

3-Octanone

13

p-Cymene

14

a

-Terpinolene

15

3-Octanole

16

Tetradecane (internal standard)

17

1-Octen-3-ol

18

Terpinen-1-ol

19

Linalool

20

2,3-Butanediol

21

b

-Caryphyllene (+ Terpinen-4-ol)

22

a

-Terpineol

23

Borneol

24

Thymol

25

Carvacrol

a

The peak numbers correspond to the numbers in Fig. 1.

Fig. 1—Typical chromatogram of volatile compounds extracted from fresh oregano L. berlandieri
leaves by a purge and trap technique. (Peak 16 = C14, tetradecane internal standard, numbers
correspond to compounds identified by GC/MS in Table 1).

Fig. 2—Effect of drying method on relative abundance of b-myrcene, a-terpinene, g-terpinene,
p-cymene, and thymol of oregano, L. berlandieri leaves. F = fresh. AD = air-dried. VMD =
vacuum-microwave- dried. FD = freeze-dried. For each compound, different letters above
the bars indicate a significant difference (p < 0.05).

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the intensity of odor (presumably due to
thymol) in vacuum-microwave and freeze-
dried samples was stronger in comparison
with air-dried samples.

The effect of a particular drying proto-

col on the release or retention of aroma
compounds is not predictable, and varies
with each herb. For example, Venskutonis
(1997) found that the five flavor volatiles
discussed above (Fig. 2) were dramatically
increased when thyme, Thymus vulgaris,
was air-dried at 60 °C but did not increase
when the herb was dried at 30 °C or freeze-
dried. Similarly,

b-myrcene, a-terpinene,

g-terpinene, p-cymene, and thymol in
sage, Salvia officinalis, showed a signifi-
cant reduction in concentration when the
plant was dried at 60 °C compared to
30 °C.

The combination of heat and atmo-

spheric oxygen in air drying facilitates en-
zymatic activity of polyphenol oxidase
(PPO) which results in the browning effect
that characterizes many air-dried food
materials (Howard and others 1996). Dur-
ing the shorter period of drying in the vac-
uum-microwave and the resulting re-
duced exposure to oxygen, less browning

was observed, as indicated by the signifi-
cantly higher ‘L’ values of vacuum-micro-
wave-dried samples (Table 2).

Dehydration processes affect the qual-

ity attribute of color to a varying degree.
While the effect of the drying protocol on
the concentration of chlorophyll was not
directly assessed in this study, color analy-
sis showed that vacuum-microwave-dried
samples were significantly greener in color
(larger negative ‘a’ values) that either air-
dried oregano or a commercially air-dried
sample of the herb. A similar effect on col-
or during vacuum-microwave and air dry-
ing was also evident in sweet basil (Yousif
and others 1999) and carrot chips (Lin and
others 1998a). These authors observed
that air-dried samples had darker hues
(smaller ‘L’ values) than vacuum-micro-
wave-dried samples.

Rehydration of plant materials is af-

fected by their polarity/hydrophobicity
and equally by other physical structures
such as the presence or absence of inter-
nal open space. The rehydration curves of
dried oregano at 30 °C and 100 °C are
shown in Fig. 3a and b, respectively. Statis-
tical analysis of the data was limited only
to the 1st two readings of the rehydration
assay (10 and 20 min at 30 °C, 0.5 and 1
min at 100 °C) because of the observation
that most rehydration in this study oc-
curred during the first 20 min of the pro-
cess, and also because of the fluctuation
of the readings past that point, most likely
as a result of the heterogeneous nature
(that is, size, thickness, age) of oregano
leaves. At low temperatures (30 °C), vacu-
um-microwave-dried oregano exhibited
significantly higher rehydration ratios as
compared to air-dried samples (Fig. 3a). At
higher temperatures, less time was re-
quired for reconstitution of all samples,
and similarly vacuum-microwave-dried
samples exhibited significantly higher re-
hydration ratios than air-dried ones (Fig.
3b). Similarly, Lin and others (1998a,b) ob-
served better rehydration of vacuum-mi-
crowave-dried carrot slices and shrimp.
Further, at high temperatures, higher re-
hydration rates occurred at the beginning
of rehydration. This was especially noted
in the vacuum-microwave and freeze-
dried samples.

In oregano, the epidermis layers are

covered by hairs or trichomes. In addition
to this surface structure, oregano plants
are known to synthesize and store their es-
sential oils in glandular type of trichomes.
These capitate glands have an unicellular
stalk and a unicellular globular to ovoid
head with the cuticle raised to form a blad-
der-like covering; they occur in depres-
sions in the epidermis. The epidermal
cells surrounding the stalk radiate to give
a very characteristic appearance to the
glands visible on the surface. The effect of
each drying method on these leaf struc-
tures was observed under the scanning
electron microscope (Fig. 4a

1,2,3

). After air

drying, oregano leaves exhibited signifi-
cant changes in their botanical structures
such as the severe shrinkage of the cuticle
and its associated structures and the un-
derlying epidermal layers (Fig. 4a

1

). Inter-

nally, the cells of both the palisade and
spongy mesophyll were extremely affect-
ed and appeared collapsed (Fig. 4b

1

). The

effect of drying on the structure of the cu-
ticle in vacuum-microwave-dried samples
was moderate as judged from the extent of
shrinkage to that layer (Fig. 4a

2

), and cells

of the epidermal and mesophyllic layers of
vacuum-microwave or freeze-dried sam-
ples had an almost identical “stretched” or
“puffed” appearance (Fig. 4b

2

and b

3

).

This is consistent with the rehydration
data that shows enhanced rehydration
rates when oregano was dried by vacuum-
microwave or freeze dryer, compared to
hot air. This ability to rehydrate at a higher
rate is likely attributed to the presence of
open structures within the leaf.

It was previously reported that the

structure of carrot (Lin and others 1998a)
and potato (Durance and Liu 1996), slices
could be puffed or expanded by vacuum-
microwave drying because the chamber
pressure was kept low during vacuum-mi-
crowave drying, and the internal pressure
within products was elevated by water va-
por. This pressure differential generates
an outward force, causing the material to
expand beyond its original dimensions,
resulting in a puffing effect; alternatively,
the low temperature that is maintained
during vacuum-microwave drying may
minimize the collapsing forces of heat dur-
ing drying. Under both circumstances the
original open structure is retained.

Table 2—HunterLab color values of dried oregano, L. berlandieri

a

Color

parameter

FD

VMD

AD

Commercial

L

51.11 ± 0.91

a

44.94 ± 1.99

b

34.34 ± 0.72

c

29.57 ± 0.96

d

a

2

6.79 ± 0.42

a

2

4.35 ± 0.57

b

2

1.04 ± 0.13

c

2.46 ± 0.34

d

b

15.37 ± 0.66

a

15.21 ± 0.81

a

10.43 ± 0.51

b

9.00 ± 0.19

c

a

For each color parameter, values (mean ± standard deviation) followed by different letters are significantly different at p<0.05

from each other FD = freeze-dried; AD = air-dried; VMD = vacuum-microwave-dried.

Fig. 3—Rehydration curves of freeze-dried
(FD), vacuum-microwave-dried (VMD), and air-
dried (AD) oregano, L. berlandieri leaves, at
(a) 30 °C or (b) 100 °C. At each time interval,
different letters above/below Y-axis values
indicate a significant difference (p < 0.05)
between treatments.

Food Chemistry and T

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Effect of Drying on Characteristics of Oregano . . .

Fig. 4—Oregano, L. berlandieri leaf. Scanning electron micrographs of: (A) epidermis x 100
and (B) cross sectional view x 480. 1 = air-dried. 2 = vacuum-microwave-dried. 3 = freeze-
dried. cut. = cuticle. c.tr. = covering trichome. c.g. = capitate gland. s = stoma. s.m = spongy
mesophyll. p.m = palisade mesophyll.

The quality of dried oregano depends

on the drying method. Vacuum-microwave
or freeze drying were found to be better

for drying the oregano plant materials
used in this study without decreasing the
concentration of its character-impact com-

pound, thymol. Unlike freeze drying, how-
ever, vacuum-microwave drying of orega-
no is a rapid process (0.4 compared to 11.5
h for air-drying) that can produce a dry
plant material with similar qualities (in
terms of color, rehydration, and structure)
to that of freeze-dried samples.

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MS 19990726 received 7/21/99; revised 1/20/00; accepted 4/

10/00.

The authors would like to acknowledge the financial sup-
port of Agriculture and Agri-Food Canada, and the Natural
Sciences and Engineering Research Council of Canada.

Authors Yousif, Scaman, and Durance are with
Food, Nutrition, and Health, Univ. of British Co-
lumbia, 6650 N. W. Marine Dr., Vancouver, B.C.
V6T 1Z4. Author Girard is with Agriculture and
Agri-Food Canada, Pacific Agri-Food Research
Center, 4200 Hwy 97, Summerland, BC, VOH 1ZO.
Address inquiries to Timothy D. Durance (E-
mail:[e-mail]).