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Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008 39
Chemical and anti-bacterial characterization of aqueous extracts of oregano,
marjoram, sage and licorice and their application in milk and labneh
A. I. Al-Turki 1, M. G. El-Ziney 2and A. M. Abdel-Salam 2*
1Plant Production and Protection Department , 2 Food Science and Human Nutrition Department, College of Agriculture and
Veterinary Medicine, Qassim University, PO BOX 6622,Buraydah 51452, Al-Qassim, Saudi Arabia.*e-mail:amsalam68@hotmail.com
Received 10 September 2007, accepted 28 November 2007.
Abstract
The antibacterial effect of aqueous extracts at a concentration of 1% (v/v) and 5% (w/w) of oregano, marjoram, sage and licorice herbs, widely used
as soft drinks and food flavors, were evaluated against E. coli and B. subtilis under in vitro conditions in tryptone soya yeast extract broth at 35°C
and in milk and labneh respectively. All tested herbal aqueous extracts demonstrated an inhibitory effect against both organisms. Oregano,
marjoram, sage and licorice extracts exhibited a higher antibacterial activity against B. subtilis compared with E. coli. Further, oregano extract had
the highest antibacterial activity against the tested bacteria compared with marjoram, licorice and sage extracts. GS-MS analysis detected a wide
range of organic and volatile compounds in herbal extracts. The inhibitory effect of some substances on the growth behavior of bacteria was also
discussed.
Key words: Oregano, marjoram, sage, licorice, GC-MS, antibacterial, milk, soft cheese.
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Journal of Food, Agriculture & Environment Vol.6(1) : 39-44. 2008
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Introduction
Microbial activity is a primary mode of deterioration of many foods
and is often responsible for the loss of quality and safety. Concern
over pathogenic and spoilage microorganisms in foods is
increasing due to the increase in outbreaks of food-borne
diseases31. Currently there is a growing interest to use natural
antibacterial compounds, like extracts of herbs and spices for the
preservation of foods, as these possess a characteristic flavor
and sometimes show antioxidant activity as well as antimicrobial
activity 30. For centuries, indigenous plants have been used in
herbal medicine for curing various diseases 10.
Studies have been reported on antimicrobial properties of
different plant parts and their extracts used as spices or aromatic
herbs including garlic, onion, cinnamon, nutmeg, curry, mustard,
black pepper, thyme, oregano, sage, rosemary, Jamaican pepper,
aniseed, basal, paprika, turmeric, bay, cardamom, cassia, Cayenne
pepper, celery, chives, clover, coriander, dill, ginger, savory and
marjoram 3, 5, 11, 12, 14, 16, 17, 19, 26, 27, 33, 37, 38. In order to reduce health
hazards, food poisoning outbreaks and subsequent economic
losses, the use of natural herbal products as green antimicrobial
substances seems to be an attracting manner to control pathogenic
microorganisms and to extend food shelf-life.
The current study was aimed to determine the constitutes of
ethanol herbal extracts of oregano, marjoram, sage and licorice by
GC-MS and to evaluate the antibacterial activities of the extracts
against Bacillus subtilis and Escherichia coli in vitro using disk
agar method, tryptone soya broth and food systems such as milk
and labneh.
Material and Methods
Herbal plant samples: The description of plants used in this
study is given in Table 1. Oregano, marjoram, sage and licorice
were collected from local market, Qassim, Saudi Arabia.
Preparation of aqueous extracts of oregano, marjoram, sage
and licorice: Aqueous extracts of oregano, marjoram, sage and
licorice were prepared using method described by Ali et al. 40
and Melendeza and Caprilesa 22. The plant material was allowed
to air dry and afterwards pulverized in a grinder. The pulverized
material 50 g was extracted with 300 ml of (80%, v/v) ethanol
(Merck-Darmstadt, Germany) in an electric blender left running
for 15 min. Afterwards, the suspension was left at room
temperature for 1 hour. The suspension was filtered twice, first
through cheese-cloth (50% cotton/50% polyester) and then
through filter paper (Whatman No.2). Ethanol was evaporated
in an oven under vacuum at 50ºC. The extracts were then filtered
and preserved in sterile dark bottles (500 ml) at -20°C until further
use.
GC-MS analysis: Approximately 20 g of herbal sample extracted
with 100 ml ethanol was directly injected into the GC-MS QP5050A
system (Model 2001, Shimadzu, Japan) equipped with auto
Table 1. Plants used in the experiments.
English name Botanical name Family Part
Oregano Origanum vulgare L. Labiataea Leaves
Marjoram Origanum majorana L. Labiataea Leaves
Sage Salvia officinalis L. Labiataea Leaves
Licorice Glycyrrhiza glabra. Fabaceae Roots
40 Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008
injector (AOC-20i, Shimadzu, Japan) and Rtx 5MS capillary column
packed with crossbond 5% diphenyl-95% dimethyl polysiloxane,
30 m x 0.25 mm, 0.25 µm film thickness (Restek capillary columns,
Hewlett-Packard, USA). Analytic conditions; temperature
program; 60°C for 8 min, rising at 4°C/min to 250°C and holding
for 10 min; injector and interface temperatures, 250 and 230°C,
respectively; carrier gas, helium at 0.8 ml/min; injection of 0.25 µl;
split ratio 1:50. The mass spectrometer was operated in the electron
impact mode using 70 eV electron energy. The mass range m/z 33-
500 was scanned. The identification of the peaks was based on
Willy and Drug spectra libraries and literature data 1.
Microorganisms: Bacillus subtilis LMQ 11 (soil isolated) and
Escherichia coli LMQ 101 (water isolated) were from the culture
collection of Laboratory of Microbiology, Qassim University.
Cultures were maintained in tryptone soya broth (TSB, Oxoid)
containing 50% glycerol at -80°C and propagated in TSB
supplemented with 0.5% yeast extract (TSYB) at 35°C for 18-h
prior to use.
Antibacterial activity of aqueous plant extracts using disk
diffusion methods: Aqueous extracts of oregano, marjoram, sage
and licorice were tested for antibacterial activity by the inhibition
zone using the disc diffusion method 39.B. subtilis LMQ 11 and
E. coli LMQ 101 were picked into nutrient broth medium, shaked
gently at 37°C for exactly 12-18 h. Nutrient agar (15 ml) was
inoculated with fresh broth culture (0.15 ml, containing 109-110
CFU/ml) and poured in sterile 9 cm Petri dishes. After evaporation
of some water in a biological safety cabinet with laminar flow
(LABCONCO Laminar Flow Products), the discs were drilled on
the agar surface and inoculated with test bacteria. Hundred
microlitres of herbal extracts or sterilized water (control) were
applied to a sterilized disc gabbing (6 mm in diameter). Then the
plates were incubated at 37°C for 18 h. The inhibition zones were
recorded in mm 5, 39. All experiments were conducted in duplicate
and the results are expressed as average values of inhibition.
The inhibitory effect of aqueous herbal extracts against B.
subtilis and E. coli cultured in TSYB: An overnight culture of B.
subtilis and E. coli was used to inoculate TSYB at level of 103-104
CFU/ml. The aqueous plant extracts were added to inoculated
broth at a concentration of 1% w/w. Controls (no extracts added)
and treatments were incubated at 35°C. Samples were drawn at
different time intervals for optical density measurement at 600 nm
(LKP-Pharmacia, Uppsala, Sweden). Growth curves were fitted
by sigmoid function using Dynamic Modeling (DMfit software)
as described by Baranyi and Roberts7.
Preparation of cheese yoghurt (labneh): Labneh (yoghurt
cheese), a kind of thickened yogurt (from Leban, Arabic for
“yogurt”), was developed as a delicious way to extend yogurt’s
shelf life and utility. Middle Easterners dip flat bread in labneh
sprinkled with chopped fresh dill, mint or thyme. It also makes a
low-fat substitute for cream cheese and sour cream in many recipes,
such as Middle Eastern Yogurt Filling and Persian Fried Eggplant
in Yogurt. It is also used to make a low-fat cheesecake, substituting
it for the cream cheese and sour cream and as a filling for blintzes.
The labneh was manufactured in the department pressing and
mangle milk protein concentrate powder (ALAPROTM, NZMP Co.,
New Zealand) was reconstituted and standardized to achieve
30% total solids contents, heated at 85°C for 30 min, cooled to
40°C and inoculated with yogurt starter composed of
Streptococcus thermophilus and Lactobacillus bulgaricus (Chr.
Hansen‘s Lab., Copenhagen Denmark). The inoculated milk was
incubated for 4-8 h at 42°C. After coagulation, the pH of the curd
was tested and stored at 4-6±2°C for further inactivation trials.
For sensory evaluation, labneh blended with herbal extracts was
judged by trained panelists for appearance, color, flavor and
overall properties according to N.A.S.A method 25.
Inactivation of B. subtilis and E. coli in reconstituted full-cream
milk and labneh: The protocol of El-Ziney and Debevere 13 of
testing antibacterial effect of inhibitory substances against
pathogens in milk and cheese was followed. In brief, an 18-h old
culture of B. subtilis and E. coli, grown in TSYB was used to
inoculate (ca. 103- 104 CFU/g) sterile reconstituted full-cream milk
(8.5% w/w), while fresh labneh was only inoculated with E. coli. A
portion of 200 g of inoculated milk and labneh was used for each
plant extract, added at a concentration of 5% (w/w). The addition
of extracts did not alter the initial pH of both milk and labneh. The
samples were placed in sterile plastic bags, and milk samples were
prepared in 250-ml conical flasks. Treated samples were incubated
at 7°C and survival of both organisms was monitored in time.
For microorganisms’ enumeration, milk and labneh duplicate
samples (10-g) were taken from each treatment at time intervals
transferred to sterile Stomacher bags (Seward, England), mixed
for 2 min with 90-g of buffered peptone saline (BPS, 0.5% peptone,
Oxoid; 0.85% NaCl) in Seward Stomacher (Seward 400, England).
Homogenized samples were serial diluted and viable cultures were
surface plated onto TSYA (Tryptone soya yeast extract agar, Oxoid)
for B. sutilis and E. coli in milk samples, meanwhile viable E. coli
in labneh samples was determined by pour plate method using
VRB-MUG agar (Oxoid).
Two trials were performed for each treatment and duplicate
samples were taken from each trial at every sampling time. Chemical
composition including protein, fat, carbohydrate and moisture of
reconstituted milk and labneh was analyzed according to official
method 2. Data were analyzed by SPSS software (Version 9, SPSS
Inc, Chicago) for the analysis of variance, the determination of
significance (P<0.05) between the treatments was using Duncan’s
multiple range test.
Results and Discussion
Compounds of herbal extracts: Ethanol extracts of oregano,
marjoram, sage and licorice were analyzed by GC-MS and results
are shown in Table 2. Fourteen compounds were identified in
oregano extract where the most represented compounds were
carvacrol (72.32%), caryophyllene (6.22%) and 1,3-pentadiene
(5.32%). The phenolic carvacrol was found to be the major
component of oregano with approximate concentration reaching
to 80% 28.
In marjoram, resorcine (25%), terpine-4-ol (23.25%),D-terpineol
(7.61%), trans-pinene (6.63%) and caryophyllene (6.28%) were
the major components. Similar results were reported by Vagi et
al. 41, further they found that extraction method would affect the
constitutes compound profile which reflects on the antimicrobial
properties. It is postulated that extracts obtained by supercritical
fluid extraction had a significant inhibitory effect higher than the
ethanolic extracts.
Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008 41
In sage extract the dominant compounds were caryophyllene
(26.1%), D-terpineol (16.98%), heptadiene-3-methyl (12.1%),
D-terpinyl acetate (11.65%), nonayene (7%) and limonene oxide
(5.24%). In contradiction with our results different compound
profile has reported by Miladinoviæ and Miladinoviæ 23 since D-
and E-thujone and camphor were detected with high
concentrations. This could be expected since the content of any
herbal extract depends on soil, climate, season and even the
extraction method. Miladinoviæ and Miladinoviæ 23 studied
antimicrobial effect of essential oil of sage from Serbia which was
extracted by hydrodistillation for 2.5 h.
Almost 15 compounds were detected in licorice with major
compounds being indend,2,3-dihydroxy-5-methoxy-1-3me
(49.58%), phenyl-4-(1-phenylethyl) naphthalene (14.95%) and
phenol,2,2-methylene (11.56%). Carvacrol was detected with
relative concentration of 2.86%.
Inhibition of B. subtilis and E. coli by herbal extracts in disk
diffusion method: The current study deals with the antibacterial
activity of some common herbs including
oregano, marjoram, sage and licorice against
undesirable bacteria such as B. subtilis and E.
coli. In general, the obtained data showed that
extracts of oregano, marjoram, sage and licorice
demonstrated antibacterial activity against
both organisms in disk diffusion method (Table
3). The mean values of the inhibition zone
diameters of oregano extract were 35 and 30
mm for B. subtilis and E. coli respectively,
while it was 30, 25 and 30 mm against B. subtilis
and 27, 20 and 25 mm against E. coli for
marjoram, sage and licorice extracts
respectively. All tested herbal extracts showed
to be more effective against Gram-positive B.
subtilis than Gram-negative E. coli bacteria.
Aqueous extracts of oregano demonstrated a
strong antibacterial activity against B. subtilis
and E. coli compared with marjoram, sage and
licorice extracts, comparable with antibiotic
tylosin. Sage extract had the lowest inhibitory
effect while marjoram and licorice extracts
exhibited the same degree of inhibition.
Inhibition of B. subtilis and E. coli by herbal
extracts in TSYB: In tryptone soya yeast
extract broth at 35°C, herbal extracts at a
concentration of 1% were able to cease the
growth of B. subtilis and E. coli (Figs 1and 2).
All herbal extracts had a bacteriostatic effect
at tested concentration. Fitting data analysis
by Baranyi and Roberts model 7 reveled that
the growth rate (µ) has decreased as a result
of herbal addition (data not shown). In the
case of B. subtilis the oregano extract (µ =
0.13) decreased the growth rate in comparing
with control (µ = 0.22) by 40%. The same trend
was noticed with marjoram, sage and licorice,
however, oregano extract was the most potent
substance.
The herbal extracts had a comparable inhibitory effect against
E. coli and led to decrease of the growth rate by 42% when
oregano extract was applied. Further, the results showed that the
rest of extracts demonstrated inhibitory effect but less than
Table 3. Antibacterial properties of oregano, marjoram, sage
and licorice extracts compared with tylosin against
B.subtilis and E. coli in disk diffusion method.
Inhibition zone diameter
(mm) against
1
Treatment
B. subtilis E. coli
Control 0 0
Oregano 35 30
Marjoram 30 27
Sage 25 20
Licorice 30 25
Tylosin antibiotic (20 µg) 40 33
1diameter of deep disc, 6 mm, included and results presented as a mean value of
three replicates.
Herbal name: 1-Oregano Herbal name: 2-Marjoram
Compound Relative ( %) Compound Relative (%)
Pyruvic acid 0.71 Trans pinene hydrate 6.63
1,3-pentadiene trimethyl 5.32 Pyruvic acid 2.44
Isoamylnitrate 3 0.14 Terpinen-4-ol 23.25
1,1-Diethyl ethene 0.27 1,1-Diethyl ethene 1.24
D- Terpineol 0.48 D-Terpineol 7.61
Triisopropyl borane 2.03 Tri-isopropyl borane 6.21
Carvacrol 72.32 Linalyl acetate 3
2-methyl 4-pentanal 0.96 1,3-Benzenediol (resorcine) 25.09
Trans caryophyllene 6.22 p-menth-8 ene-2-ol 3.42
5-Nitro m-xylene 4.4 4-pentanal-2-methyl 4.89
4-Tertbutyl catechine 2.24 Trans caryophyllene 6.28
Longipinene epoxide 1.67 Allyl ether 2.62
E-Ocimene 0.55 Not identified 7.32
Allyl ether 0.96
Not identified 1.73
Total 100 Total 100
Herbal name: 3-Sage Herbal name: 4-Licorice
Compound Relative (%) Compound Relative (%)
2,5-Octadiene 2.17 Glycerin 0.32
1,6-Octadiene 2.06 Resorcinol 2.28
1,6-Heptadiene-3-methyl 12.07 Carvacrol 2.86
1-Nonayene 7 2,4-Ditertbutylphenol 0.47
D-Terpineol 16.98 P-Tertbutylacetophenone 3.49
Linalyl acetate 1.5 Acetophenone,2,4,5-triethyl 0.25
D-Terpinyl acetate 11.65 4-Hydroxyphenylacetic acid 0.41
Allylmethallyl ether 4.01 Phenol,2,2-methylene 11.56
Trans caryophyllene 26.07 Carpachromene 1.6
D-Humulene 3.92 Coumaran 2.27
Veridiflorol 3.77 Di(pentamethylphenyl l-ketone) 3.02
Limonene dioxide 5.24 Decamethylbenzophenone 2.12
Not identified 3.56
Phenol,2(3,4-dihydro-8-methyl-
8-(4-methyl) 4.82
Phenyl-4-(1-
phenylethyl)naphthalene 14.95
Indend,2,3-dihydroxy-5-
methoxy-1-3me 49.58
Total 100 Total 100
Table 2. The chemical constitutions of oregano, marjoram, sage and licorice extracts
analyzed and detected by gas chromatography-mass spectrum.
42 Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008
oregano extract. In agreement with other reports, the present
study revealed that Gram-negative bacteria are less susceptible
than Gram-positive bacteria 9 since the former posses an outer
membrane surrounding the cell wall which restricts diffusion of
hydrophobic compounds through its lipopolysaccharide
covering 36.
Inhibitory effect of oregano, marjoram, sage and licorice extract
application in milk and labneh: The chemical composition of
milk and labneh used throughout the present study is shown in
Table 4. Sensory evaluation of labneh blended with oregano,
marjoram, sage and licorice extracts is shown in Table 5.
The antibacterial activity of herbal aqueous extracts of oregano,
marjoram, sage and licorice against undesirable bacteria B. subtilis
and E. coli in milk and labneh are shown in Figs 3, 4 and 5. In the
present study, in food model systems, to achieve a bactericidal
effect of herbal extracts a greater concentration (5%) is required
compared to the amount used in TSYB (1%), however, raising the
concentration to 10% was able to enhance the killing effect (data
not shown). The concentration used in food application could
vary from 2 to 100 fold of that used in the in vitro assays depending
on the herb type and food system used 20, 35.
In milk and in the absence of herbal extract the number of viable
B. subtilis and E. coli tended to be stable within 4 days at 7°C.
The addition of herbal extracts led to reduce the viability of both
organisms. The reduction in the number of B. subtilis was more
pronounced (P> 0.05) compared to the culture of E. coli. When
oregano extract was applied the bacilli decreased by 2.5 log cycle
after 1 days while Escherichia was reduced only by 0.5 log cycle
during the same time. At Day 4, the oregano extract was able to
diminish B. subtilis to non-detectable limit while E. coli decreased
by 2.5 log cycle (Figs 4 and 5). Marjoram, sage and licorice extracts
had an inhibitory effect but less extended compared with oregano
extract. The three herbal extracts having same category, showed a
reduction rate amounted to # 2.5 log cycle against B. subtilis after
2 days (Fig. 3). Total destruction was reached after 3 days for sage
and licorice extracts where it was at Day 4 with marjoram extract.
Less decline rate was noticed against E. coli and the order was:
sage > licorice = marjoram.
In labneh, total solids and milk fat contents reached to 30.55%
and 18%, respectively, and herbal extracts demonstrated
bactericidal effects against E. coli at 7°C (Fig. 5). Extracts exerted
the same trend of inhibitory effect exhibited in milk, as oregano
extract had the most potent effect followed by sage. Meanwhile,
in labneh the destruction rate was less pronounced compared
with the effect in milk. This reflects that high fat and protein
contents offer a good protection background for microorganisms
which naturalizes the inhibitory effect of antimicrobial substances5.
In the present study, the pH of labneh was 5.8 and it is believed
that lowering the pH to 5 will enhance the inhibitory effect of
herbal extracts 42.
Carvacrol is an essential compound of oregano extract 4 and its
antimicrobial effect relies on a system of delocalized electrons
and hydroxyl group on the phenolic ring 34. The compound causes
a bacterial death by damaging their cytoplasmic membrane
integrity, affects pH homeostasis and equilibrium of inorganic
ions 21 and leads to the collapse of the proton motive force and
depletion of the ATP pool 34. Burt 8 stated that in view of published
data on the antimicrobial effect of essential oils in food, the
following approximate general ranking can be made: oregano>
thyme> mint> rosmary> sage.
Oregano, marjoram and sage have been used in folk medicine to
treat colds, coughs, gastrointestinal problems and a variety of
other conditions, and several plants in the genus reportedly have
antibacterial, antifungal and antimicrobial properties due to the
phenol carvacrol 18, 32. The crude extract of licorice has also found
commercial use as a food additive in Japan, as it contains the
sweetening principal glycyrrhizin 29. Chemical investigations have
revealed the presence of a wide variety of bioactive phenolic
constituents in licorice 29. Licorice flavonoids were found to have
antimicrobial activity against methicillin-resistant Staphylococcus
aureus 15.
The sensory data from three trials were aggregated for analysis.
In general, the labneh’s produced with herbal extracts were
accepted by the panelists (Table 5). With exception to the color of
labneh’s, the rest of the sensorial properties, appearance, flavor
and overall acceptability, showed no significant difference
between labneh’s produced with oregano and marjoram extracts
compared to the control labneh (no extract was added) and
preferred over those produced with sage and licorice.
Generally, all of tested herbal extracts had inhibitory effects
against tested undesirable bacteria. It is concluded from the
obtained results that extracts of oregano and sage and to a less
extent marjoram and licorice could be applied successfully to
control food-borne pathogens in milk and soft cheese, labneh.
The combination of different herbal extracts would be suggested
for further research, the possible synergistic effects would
enhance the bactericidal effect.
Table 4. Chemical composition of milk and labneh used in this
study (g/100 g).
Ingredient Carbohydrate Fat Protein Ash Total solids
Milk 3.25 2.26 2.50 0.51 8.52
Labneh 6.50 18.00 5.0 1.05 30.55
Sensorial
characteristics
Oregano labneh Marjoram labneh Sage labneh Licorice labneh Plain labneh
(control)
Appearance +++ +++ ++ ++ +++
Color ++ ++ ++ ++ +++
Flavor +++ +++ ++ ++ +++
Overall +++ +++ ++ ++ +++
(+++) very good (++) good (+) accepted ( - ) unaccepted
Table 5. Sensory evaluation properties of labneh blended with oregano, marjoram, sage and licorice extracts.
Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008 43
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44 Journal of Food, Agriculture & Environment, Vol.6 (1), January 2008
Figure 1. The antibacterial activity of oregano, marjoram, sage and
licorice extracts at a concentration of 1% (v/v) against B. subtilis in
TSYB media at 37ºC.
0
0.2
0.4
0.6
0.8
1
01234567
control
Oregano
Marjoram
Sage
Licorice
Time (h)
OD 600 nm
0
1
2
3
4
5
01234
control
Oregano
Marjoram
Sage
Licorice
Figure 3. The effect of oregano, marjoram, sage and licorice extracts at
a concentration of 5% (v/w) on the survival of B. subtilis in reconstituted
milk (T.S; 8.5%) at 7ºC.
Time (d)
Log CF U/g
Figure 5. The effect of oregano, marjoram, sage and licorice extracts
at a concentration of 5% (v/w) on the survival of E. coli in labneh at
7ºC.
2
.5
3
.5
4
01234567
control
Oregano
Marjoram
Sage
Licorice
Time (d)
3
2
Log CF U/g
Figure 4. The effect of oregano, marjoram, sage and licorice extracts at a
concentration of 5% (v/w) on the survival of E. coli in reconstituted milk
(T.S; 8.5%) at 7ºC.
0
1
2
3
4
5
6
01234
control
Oregano
Marjoram
Sage
Licorice
Time (d)
Log CF U/g
Figure 2. The antibacterial activity of oregano, marjoram, sage and
licorice extracts at a concentration of 1% (v/v) against E. coli in TSYB
media at 37ºC.
0
0.2
0.4
0.6
0.8
1
1.2
012345678
OD 600nm
control
Oregano
Marjoram
Sage
Licorice
OD 600 nm
Time (h)
