1744

Journal of Food Protection, Vol. 72, No. 8, 2009, Pages 1744–1752

Copyright G, International Association for Food Protection

Research Note

In Vitro Antimicrobial and Antioxidant Activity of Commercial

Rosemary Extract Formulations
ANJA KLANČNIK,1 BERNARDA GUZEJ,1 MAJDA HADOLIN KOLAR,2 HELENA ABRAMOVIČ,1 AND
SONJA SMOLE MOŽINA1*

1Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia; and
2Vitiva, Ltd., Nova Vas pri Markovcih, SI-2281 Markovci, Slovenia

MS 08-524: Received 17 October 2008/Accepted 16 March 2009

ABSTRACT
Phenolic plant extracts are sources of natural bioactive compounds, which can inhibit the rate of food spoilage. MIC and
MBC concentrations of four oil- or water-soluble rosemary (Rosmarinus officinalis) extracts against gram-positive (Bacillus and
Staphylococcus) and gram-negative (Campylobacter and Salmonella) bacteria were determined by using disk diffusion, agar
dilution, and broth microdilution methods, as well as bacterial survival kinetics in a macrodilution test. To describe the
antioxidant properties of the extracts, the reducing power, free radical scavenging effectiveness, and b-carotene bleaching test
were used. The antimicrobial and antioxidant activity depended on the concentration and chemical nature of the phenolic
compounds in the extracts. Gram-positive bacteria were more sensitive than were gram-negative bacteria, especially for oil-
soluble extracts with carnosic acid as the major phenolic compound. A microdilution method based on ATP measurement was
found to be a useful, rapid technique for determining antibacterial efficiency, and its results correlated well with MICs from
survival curve measurement. Reducing power and free radical scavenging effectiveness was higher in water-soluble formulations,
according to their higher total phenolic content, but in an aqueous emulsion system of linoleic acid, they exhibited lower
antioxidant activity. This correlated well with the higher efficiency of antimicrobial activity of oil-soluble formulations, despite
the lower total phenolic content of these extracts.

Phenolic plant extracts may possess antimicrobial and
antioxidant activity that can inhibit or delay both oxidative
and microbiological food spoilage (16, 52, 57, 62, 63). To
prevent growth of undesirable microorganisms, spoilage,
off-flavor, rancidity, and deterioration and thus improve
stability and prolong shelf life, extracts should display a
wide spectrum of antimicrobial and antioxidant activities
because food products are seldom contaminated by one
species or spoiled for a single reason (25). Biological
activity of phenolic plant extracts, including antimicrobial
and antioxidant activity, is influenced by plant growth and
harvest conditions, as well as by extraction methods, which
give extracts of different chemical composition. Extracts
with similar concentrations of total phenolics may vary
remarkably in their antioxidant and antimicrobial activity (4,
7, 9, 16, 29, 33, 49, 54, 60).
Rosemary (Rosmarinus officinalis L.) is an aromatic
plant and thus a flavoring agent, widely used in foods. Its
extracts have been introduced as preservatives in the food
industry (24). Rosemary extract formulations are the only
ones commercially available for use as antioxidants in the
European Union and the United States, and they are
marketed in an oil-soluble form, as a dry powder, and in
* Author for correspondence. Tel: +386 1 423 11 61; Fax: +386 1 256 62 96;
E-mail: Sonja.smole@bf.uni-lj.si.
water-dispersible or water-miscible formulations (1, 5). The
nonnutrient secondary metabolites of rosemary such as the
phenolic diterpenes, carnosol, carnosic acid, methyl carno-
sate, rosmanol, and epirosmanol, and phenolic acids such as
ferulic, rosmarinic, and chlorogenic and caffeic acids, have
already been reported to possess diverse biological
activities, including antioxidant and antimicrobial activity
(8, 13, 17, 23, 28, 34, 40, 46, 59).
There is great interest in plant antimicrobials, and
several methods are available to detect their inhibitory
activity. Various publications have documented the antimi-
crobial activity of plant extracts including rosemary. Several
methods have been used, and they are based on different
principles. So the results obtained are influenced by the
method selected, by the microorganism or specific strain
used, and by the plant extract test compound (with different
phenolic compounds) and degree of solubility (2, 15, 16, 32,
47, 56), making their comparison problematic.
The antioxidant activity of phenolic compounds has
been attributed to various mechanisms, and numerous
techniques are available to evaluate the antioxidant
properties of these compounds. Some methods, such as
free radical scavenging assays, might provide information
on the capability of an antioxidant to prevent reactive radical
species from reaching lipoproteins, polyunsaturated fatty
acids, DNA, amino acids, proteins, and sugars in biological
J. Food Prot., Vol. 72, No. 8 ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACTS
TABLE 1. The contents of total phenolic compounds and of carnosic and rosmarinic acids, as well as CR, EC50, and CAA valuesa
Total phenolic content Carnosic acid Rosmarinic acid
Plant extract (mg/g) (mg/g) (mg/g)
V15 198 156 6 8 —b
V40 438 407 6 5 — 66.1
A15 593 — 161 6 4
A40 915 — 420 6 6
a
The contents of total phenolic compounds are expressed as chlorogenic acid equivalents. CR, reducing power; EC50, concentration of
rosemary extracts in the reaction mixture that causes a decrease in the initial DPPH? concentration by 50%; CAA, antioxidant activity
coefficient in an aqueous emulsion of linoleic acid and b-carotene.
b
—, not present.
and food systems (27). In recent times, the capability of
rosemary extracts to scavenge free radicals was investigated
by Almela et al. (3), Nogala-Kalucka et al. (37), Moreno et
al. (34), and Yesil Celiktas et al. (61). In the evaluation of
antioxidants, in addition to their free radical scavenging
effectiveness, it is important to consider other factors,
including interfacial phenomena affecting their partition
behavior in multiphase systems. Some investigators (10, 11)
determined the effectiveness of rosemary extracts on free
radicals formed in an aqueous emulsion system of linoleic
acid and b-carotene. The iron-reduction capacity of
rosemary extracts has also been reported (21). Investigations
that evaluated the antioxidant activity of rosemary extracts
by a combination of different methods and correlation of
antioxidant properties to the antimicrobial activity are scarce
(5, 34).
The objective of this work was to examine the in vitro
antimicrobial and antioxidant activity of four oil- or water-
soluble rosemary extract formulations and to interpret them
in terms of their composition. The aim was to determine and
to compare their MICs and MBCs against gram-positive
(Staphylococcus aureus and Bacillus cereus) and gram-
negative (Campylobacter jejuni and Salmonella Infantis)
bacteria, with different methods, and to describe the kinetics
of survival and/or inactivation. The reducing power, the free
radical scavenging effectiveness, and the b-carotene bleach-
ing test were selected to describe the antioxidant potential of
the extracts.
MATERIALS AND METHODS
Plant extracts formulations and pure substances. Com-
mercial rosemary extract formulations V15, V40, A15, and A40
were supplied from Vitiva d.d. (Markovci, Slovenia). According to
the product specification (using a high-performance liquid
chromatography method), the main active phenolic compounds
in oil-soluble (V15 and V40) and water-soluble (A15 and A40)
formulations were carnosic and rosmarinic acid, respectively
(Table 1). The activity of the pure substances carnosic and
rosmarinic acid (Chromadex, Santa Ana, CA) was tested as well.
Reagents and solvents. The following reagents were
obtained from Merck (Darmstadt, Germany): chloroform (analyt-
ical grade), ethanol (96%), sodium carbonate (analytical grade),
and trichloroacetic acid (99.5%). b-Carotene (.97%), 2,2-
diphenyl-2-picrylhydrazyl (DPPH?) reagent, Folin-Ciocalteu re-
agent, chlorogenic acid (.95%), linoleic acid (.95%), Tween 20,
1745
21
CR (mg/ml) EC50 (mg/ml) CAA
27.2 6 0.4 0.0227 6 0.0005 0.59 6 0.01
6 0.6 0.0092 6 0.0002 0.79 6 0.02
69.5 6 0.3 0.0132 6 0.0002 0.12 6 0.02
124 6 1 0.0074 6 0.0001 0.34 6 0.01
and nalidixic acid ($98%) were purchased from Sigma-Aldrich
GmbH (Steinheim, Germany). Potassium ferricyanide (analytical
grade) was obtained from Kemika (Zagreb, Croatia). Ferric trichloride
(.99%) was obtained from Carlo Erba Reagenti (Rodano, Italy). The
phosphate buffer was made by disodium hydrogen phosphate
(analytical grade) purchased from Zorka (Šabac, Croatia), and
potassium hydrogen phosphate (analytical grade) was purchased
from Kemika. BacTiter-Glo reagent (Promega Corp., Madison, WI)
and oxytetracycline solution (Krka, Novo mesto, Slovenia) were also
used. Water was prepared by purification with a Milli-Q water
purification system (Millipore, Bedford, MA).
Determination of total phenolic content. The total content
of phenolic compounds in the extracts was determined
spectrophotometrically with Folin-Ciocalteu reagent, using a
slightly modified method by Gutfinger (26). The reaction
mixture contained 200 ml of rosemary extract diluted in 96%
ethanol, 125 ml of freshly prepared Folin-Ciocalteu reagent, and
125 ml of 20% sodium carbonate solution. The final mixture
was diluted to 1 ml with deionized water. The mixture was kept
in the dark at ambient temperature for 40 min to complete the
reaction, and then the absorbance at 765 nm was measured on a
model 8453 UV-visible spectrophotometer (Hewlett Packard,
Waldbronn, Germany) with a 1-cm cell. Results are expressed
as milligrams of chlorogenic acid per gram of extract. The
reaction was conducted in triplicate, and the results were
averaged.
Bacterial strains, culture media, and growth conditions.
Antimicrobial effects were individually tested against Bacillus
cereus WSBC 10530 (clinical isolate), Staphylococcus aureus
ATCC 25923 (clinical isolate), Campylobacter jejuni ATCC
33560 (bovine feces isolate), and Salmonella Infantis ŽM9 (poultry
meat isolate). B. cereus, S. aureus, and Salmonella Infantis were
incubated aerobically at 37uC in Mueller-Hinton broth (MHB) or
agar (MHA; Oxoid, Ltd., Basingstoke, UK), while C. jejuni was
incubated microaerobically at 42uC in MHB with defibrinated
horse blood (Oxoid, Ltd.) added. For inocula preparation, all
bacteria were incubated for 20 h in MHB and for antibacterial
activity assays 1 ml of each, appropriately diluted (in MHB) to ca.
106 CFU/ml, was used.
Antibacterial activity. The disk diffusion, agar dilution, and
broth microdilution methods were used for determination of MICs
(MICdif, MICdil, and MICmdil, respectively). Bacterial survival
curves were followed to determine MICs and MBCs, with the
broth macrodilution method. Lyophilized plant extracts V15 and
V40 were dissolved in 96% ethanol to prepare stock solutions at a
concentration of 10 mg/ml and further diluted in MHB to a
1746 KLANČNIK ET AL.
working solution of 1.25 mg/ml or lower. For lyophilized plant
extracts A15 and A40, stock solutions with a concentration of
160 mg/ml and a working solution of 20 mg/ml or lower were
used. When necessary, stock solutions with higher concentrations
were used and further diluted in a similar way. Measurement for
each antimicrobial method was repeated at least in triplicate, and
the most representative values were used.
Disk diffusion method. For the disk diffusion assay (12),
1 ml of each microbial suspension was uniformly spread on a
sterile petri dish with MHA for B. cereus, S. aureus, and
Salmonella Infantis and on MHA with blood for C. jejuni. A
sterile, blank paper disk (6 mm in diameter; Difco, Becton
Dickinson, Sparks, MD) was placed on the surface of each agar
plate and was impregnated with 10 ml of each prepared plant
extract serially diluted in MHB. Using the disk diffusion method,
almost all possible concentrations were tested: for both gram-
positive bacteria in the range of 0.313 to 20.0 mg/ml and for both
gram-negative bacteria in the range of 20.0 to 100.0 mg/ml. Plates
were incubated for 24 h as stated above. Antibacterial activity as
MICdif was determined as the lowest concentration that produced
any inhibition zone around a disk after the 24 h of incubation. The
disks impregnated with sterile distilled water and ethanol served as
negative controls, and a disk with antibiotic (oxytetracycline or
nalidixic acid) served as a positive control.
Agar dilution method. For the agar dilution method, twofold
serial dilutions of plant extracts or pure phenolic acids were made
in liquid MHA media to obtain desired final concentrations and the
agar was allowed to solidify. Bacteria were inoculated as described
above for the agar diffusion method. Concentrations used for gram-
positive bacteria B. cereus and S. aureus ranged from 0.020 to
1.25 mg/ml, and for gram-negative bacteria C. jejuni and
Salmonella Infantis, from 1.25 to 10.0 mg/ml. The MICdil was
the lowest concentration where no growth was observed after 24 or
48 h. Negative controls included ethanol, in amounts correspond-
ing to the highest quantity present in the test. Inoculated agar plates
without extract added served as positive controls.
Broth microdilution method. For the broth microdilution
test bacterial culture (50 ml) in the early stationary phase (ca.
106 CFU/ml) was added to the wells of a sterile 96-well microtiter
plate containing 50 ml of twofold serially diluted plant extracts in
MHB. Concentrations ranged from 5.0 to 0.020 mg/ml. The final
volume in each well was 100 ml. Control wells were prepared with
culture medium, bacterial suspension only, plant extracts only, and
ethanol in amounts corresponding to the highest quantity present.
Contents of each well were mixed on a microplate shaker
(Eppendorf, Hamburg, Germany) at 750 rpm for 1 min prior to
incubation for 24 h, as described above. The MICmdil was the
lowest concentration where no metabolic activity was observed
after 24 h on the basis of the absence of a bioluminescence signal,
measured with a Microplate Reader (Tecan, Mannedorf/Zurich,
Switzerland) after adding 100 ml per well of BacTiter-Glo reagent
and 5 min of incubation in the dark, following the manufacturer’s
instructions (43).
Kinetics of inactivation by using the broth macrodilution
method. For the broth macrodilution method, the appropriate
volume of extracts was added to 5 ml of growth medium to give a
final concentration according to the results of previous agar
diffusion, agar dilution, and broth microdilution methods in the
range of 0.020 to 100 mg/ml, depending on the culture tested. The
diluted cultures were inoculated, shaken, and incubated as
J. Food Prot., Vol. 72, No. 8
described above. Controls were tested as described above.
Bacterial survival was followed by taking samples at 0, 3, 6, 9,
and 24 h and plating on MHA after serial sample dilutions and
incubation for 24 h. The MIC was the lowest concentration that
inhibited bacterial growth, and the MBC was the lowest
concentration that resulted in nondetectable cells after 24 h of
incubation. All experiments were independently repeated three or
more times, and the mean log CFU per milliliter as well as standard
deviations were calculated.
Reducing power. The reducing power of rosemary extracts
was determined according to Juntachote et al. (30). Half a milliliter
of solution of rosemary extract at different concentrations was
mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml
of potassium ferricyanide (1%). After 20 min, the reaction was
terminated by adding 2.5 ml of trichloroacetic acid solution (10%).
The mixture was centrifuged for 15 min. The supernatant (2.5 ml)
was mixed with 2.5 ml of water and 1.0 ml of ferric trichloride
solution (1%). The absorbance was measured on a model 8453
Hewlett Packard UV-Visible spectrophotometer (Hewlett Packard,
Waldbronn, Germany) with a 1-cm cell at 740 nm, against ethanol
as a blank. Each measurement was conducted in triplicate, and the
results were averaged.
Free radical scavenging effectiveness (DPPH? assay). The
free radical scavenging effectiveness of rosemary extracts was
determined according to Brand-Williams et al. (6). A solution of
DPPH? in ethanol was added to the solution of rosemary extract at
different concentrations. Then the absorbance at 517 nm was
measured at t 5 30 min against ethanol as a blank. The control
solution consisted of DPPH? in ethanol. The remaining level of
DPPH? was calculated by using the following equation:
 
As
%%ofofremaining DDPH? ~ 100 |
remainingDDPH ð1Þ
Ac
where As is the absorbance of the sample, and Ac is the absorbance
of the control. Triplicate measurements were conducted, and the
results were averaged.
b-Carotene bleaching method. The antioxidant activity of
rosemary extracts in an aqueous emulsion system of linoleic acid
and b-carotene was determined according to Moure et al. (35). The
b-carotene in chloroform (2 ml) was mixed with 40 mg of linoleic
acid and 400 mg of Tween 40. After evaporation of chloroform,
100 ml of oxygenated distilled water was added and mixed.
Aliquots of 5.0 ml of this emulsion and a 0.2-ml solution of
rosemary extract were mixed. As a control, a mixture of 0.2 ml of
ethanol and 5.0 ml of the above emulsion was used. The
absorbance was measured at 470 nm, immediately after prepara-
tion (time [t] 5 0 min) and after incubation at 50uC at t 5 80 min
against the blank. The blank was prepared by adding 0.2 ml of
ethanol to an emulsion consisting of linoleic acid, Tween 40, and
distilled water. The antioxidant activity coefficient, CAA, was
calculated according to the following equation:
 
Asðt~0Þ { Asðt~80Þ
CAA ~ 1 { ð2Þ
Acðt~0Þ {Acðt~80Þ
where At(t50) and Ac(t50) are the initial absorbance of the sample
and of the control, respectively. As(t580) and Ac(t580) are the
absorbance at t 5 80 min of the sample and of the control,
respectively. Each measurement was repeated in triplicate, and the
results were averaged.
Statistical analysis. For the results in Table 1 presenting
carnosic and rosemary acid content, Statgraphics Centurion XV
J. Food Prot., Vol. 72, No. 8 ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACTS 1747

TABLE 2. Antimicrobial activity of plant extracts and pure phenolic acids expressed as MIC, determined by disk diffusion, agar dilution, and microdilution tests, based on ATP measurement and

macrodilution
(Statgraphics Co., Tulsa, OK) was used for statistical analysis. All

MBC

20.0
20.0
20.0
20.0
10.0
5.0
Broth
measurements of MICs were repeated at least in triplicate, and the
most representative values were used. The results for survival curves

MIC

5.0
5.0
2.5
2.5
5.0
2.5
and antioxidant measurements were independently repeated three or

Salmonella Infantis ŽM 9

more times and expressed as means with standard deviation. The
least-squares method was used to perform regression analysis.

MICmdil
dilution
Micro-

5.0
5.0
2.5
2.5
5.0
2.5
RESULTS AND DISCUSSION
Antimicrobial activity. The effect of rosemary extracts

diffusion dilution
MICdil
Agar

1.25
9.0
9.0
9.0
8.0
5.0
was examined against the foodborne pathogenic bacteria S.
aureus, B. cereus, C. jejuni, and Salmonella Infantis. Gram-

.100.0
100.0
100.0
100.0
MICdif
positive strains were much more sensitive to rosemary

Disk

ND
ND
extracts. For example, by the agar dilution method, the
MICdil for S. aureus and B. cereus was 0.078 to 5.0 mg/ml,

0.313

0.313
MBC
macrodilution
whereas for Salmonella, it was 5.0 to 10.0 mg/ml (Table 2).

2.5

5.0
5.0

2.5
Broth
Campylobacter seemed to be quite sensitive to the various

Campylobacter jejuni ATCC 33560

extracts tested, which is consistent with earlier reports (18,

0.156

0.156
MIC

1.25

1.25
2.5
2.5
45, 51, 53). S. aureus has been reported to be one of the
most sensitive bacteria to rosemary extracts (48). However,
comparing the MICs in Table 2, B. cereus was even more

MICmdil
dilution
Micro-

0.156

0.156
1.25

1.25
2.5
2.5
susceptible than was S. aureus. The gram-negative Salmo-
nella exhibited marked resistance to the extract formulations
tested. The outer membrane surrounding the cell wall in

diffusion dilution
MICdil

1.25
Agar

8.0
7.0
10.0
5.0
5.0
gram-negative bacteria, which restricts diffusion of com-
pounds through its lipopolysaccharide covering, may be the
reason for this resistance (19, 55). The single membrane of

20.0
40.0

.40.0
.40.0
MICdif
Disk

ND
ND
gram-positive bacteria is considerably more susceptible to
permeation by antimicrobials. The sensitivity of bacteria to

0.156
0.156

0.156
MBC
polyphenols also depends on the bacterial species and
macrodilution

5.0
5.0

5.0
polyphenol structure (44, 53, 60). This was significant for
Broth
Staphylococcus aureus ATCC 25923

the bacterium Campylobacter, which has survival strategies

0.156
0.156

0.156
MIC

5.0
5.0

2.5
different from other foodborne bacteria, because it lacks
stress adaptive responses described in other enteropathogens
(36, 38, 39).
MICmdil
dilution
Micro-

0.156
0.156

0.156
For preliminary screening of antibacterial activity, the 5.0
5.0

5.0
disk diffusion method was used. However, some extracts
showed very low efficiency. Water-soluble (A15 and A40)
broth macrodilution tests with measurement of the kinetics of inactivation

0.625
0.156

0.156
diffusion dilution
MBC MICdif MICdil
Agar

formulations gave a minimal inhibition zone against gram-

5.0
5.0

10.0
negative C. jejuni and Salmonella Infantis only at
0.625

concentrations of 40.0 and 100.0 mg/ml, respectively

Disk

ND
ND
5.0

20.0
20.0

(Table 2). As reported by Moreno et al. (34), the absence

of an inhibition zone does not necessarily mean an inactive
macrodilution

—a
—
—
—
—
—

compound. Compounds less polar diffuse more slowly into

Broth

the culture medium. The diameter of the inhibition zone is

0.156
0.078

0.078
Bacillus cereus WSBC 10530

MIC

influenced by the rate of diffusion of the antimicrobial agent

1.25
2.5

5.0

through the agar, and the hydrophobic nature of most plant

extracts prevents uniform diffusion of these substances
MICmdil
dilution
Micro-

0.156
0.078

0.078

through agar media (20, 47). Thus, a quantitative determi-

1.25
2.5

5.0

nation of antibacterial activity was made through the agar

dilution method, where activity was shown at lower
diffusion dilution

concentrations compared with the disk diffusion method

MIC (mg/ml) MICdif MICdil

0.156
0.078

0.156
Agar

1.25
5.0

6.0

(Table 2). Oil-soluble formulations (V15 and V40) inhibited

growth of B. cereus and S. aureus at 0.078 to 0.625 mg/ml,
0.625
0.313

Carnosic acid NDb

Disk

ND

—, not achieved.
ND, not defined.

while water-soluble formulations (A15 and A40) required

5.0
20.0

1.25 to 5.0 mg/ml.

Since the agar dilution method is time- and material-
Rosmarinic

consuming, we introduced the broth microdilution method

acid

as a rapid quantitative determination of MIC based on

V15
V40
A15
A40

measurement of the bioluminescent signal, which is

a
b
1748 KLANČNIK ET AL.
FIGURE 1. Staphylococcus aureus sur-
vival curve during exposure to V40 and
A40. Each point represents log of the mean
6 standard deviation CFU per milliliter.
proportional to the amount of ATP present. Using this
microdilution method, the antibacterial activity determined
as MICmdil was at the same or lower concentrations
compared with the agar dilution method (MICdil) (Table 2).
The results in Table 2 show that the MICs obtained by
different methods differed significantly. This was the reason
that we evaluated the inhibitory or inactivation efficiency of
rosemary extracts during 24 h of incubation by using the
broth macrodilution method in liquid media at concentra-
tions found to be inhibitory by the diffusion and dilution
tests (Figs. 1 through 4). In some cases, higher concentra-
tions were required for inhibition (V40 and A40 extracts).
At 24 h, inhibition of growth was found at a concentration
lower than that shown with agar screening tests, but the
same as for the broth microdilution test. One notable
difference was found with the V40 and A40 against S.
aureus, but regardless of the extract, the number of cells
decreased immediately after extract exposure (Fig. 1).
While V40 significantly inhibited survival, and the bacterial
number correlated to exposure time, exposure to A40
caused interesting response variability. These results
showed that S. aureus cells could possibly adapt their
mechanism, but cannot remain viable for extended periods
in the presence of rosemary extracts. As shown in Figure 1,
the concentration determined with dilution methods is
inhibitory, and at the same time bactericidal, for S. aureus
culture. The results in Figure 2 show the activity of V40 and
A40 extracts against C. jejuni. Compared with the results in
Table 2, both extracts showed a stronger antimicrobial
FIGURE 2. Campylobacter jejuni survival
curve during exposure to V40 and A40.
Each point represents log of the mean 6
standard deviation CFU per milliliter.
J. Food Prot., Vol. 72, No. 8
effect on C. jejuni cells in liquid media by the broth
macrodilution and microdilution methods, probably indicat-
ing weaker diffusion or other antagonistic effects of the
tested substances in campylobacter solid medium and
consequently higher MICs in agar. When exposed to
concentrations determined as MICdif and MICdil for both
extracts, the cell number was dramatically reduced under the
detection limit immediately or within first 6 h. The cells
were destroyed during 24-h incubation periods and at lower
concentrations, such as 5.0 mg/ml for A40 and interestingly
at 0.625 mg/ml for V40, indicating the bactericidal effect
(MBC). An inhibition effect on C. jejuni was also found at
MICmdil for both formulations tested (Fig. 2). According to
this, we can assume that simple screening of MICs on agar
media does not necessarily give reliable decision-making
results. Following the kinetics of inactivation in liquid
media over 24 h of incubation, quick inactivation was
confirmed at the MICdif and thus differences among the
activities of the agents tested at concentrations determined
as MICdif and MICdil. Further characterization by using the
microplate method showed a more accurate range of
antibacterial efficiency, which also correlated with the
MIC assessed from Salmonella Infantis survival curves
(Fig. 3). The concentration predetermined as MICdif de-
stroyed the Salmonella Infantis immediately after 100.0 mg/
ml extract addition, while 20.0 mg/ml was determined as
MBC (Fig. 3). Interestingly, our results show that the
kinetics of inactivation over the entire period of exposure to
both extracts were comparable. This was visible also in
J. Food Prot., Vol. 72, No. 8 ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACTS
Figure 4, where the results provide another example of
growth inhibition of B. cereus by extracts V40 and A40
achieved at very low concentrations. All concentrations
tested inhibited growth, but culturable cell reduction was
only minor; thus, significant differences among the kinetics
of antimicrobial activity at MICdif, MICdil, and MICmdil
(Fig. 4) were not confirmed. These results indicate the
possible influence of sporulation as part of the adaptation
and survival mechanism during extract treatment. Vegeta-
tive cells are more susceptible to plant extracts than are
spores, which can adapt and survive extract treatment stress.
Thus, we confirmed different sensitivity to antimicrobials as
reported for cells and spores (58).
Only for the gram-positive, nonsporogenic bacterium
Staphylococcus do endpoint agar dilution and broth dilution
methods seem to give similar concentrations (MICdil,
MICmdil, and MIC), which are inhibitory and at the same
time bactericidal (MBC), as demonstrated by the measure-
ment of kinetics of inactivation. In gram-negative bacteria
Campylobacter and Salmonella, MICmdil was comparable to
the antibacterial effect presented as MIC, assessed from the
kinetics of inactivation curves (Table 2). From the results
presented, we can conclude that the microdilution method
based on measurement of the metabolic indicator ATP is
more sensitive than are screening agar methods and
consequently, is most appropriate for rapid quantitative
determination of antimicrobial activity. However, further
work on cultures treated with rosemary extracts is needed
to more accurately determine the extract effect on
1749
FIGURE 3. Salmonella Infantis survival
curve during exposure to V40 and A40.
Each point represents log of the mean 6
standard deviation CFU per milliliter.
inactivation of spores and to clarify the mechanisms of cell
growth inhibition.
Carnosic acid and rosmarinic acid are the main
bioactive antimicrobial compounds present in the rosemary
extracts (34, 41). The tested rosemary extracts differ in the
content of total phenolics expressed as chlorogenic acid
equivalents in the extracts determined according to Folin-
Ciocalteu and also in the relative amounts of carnosic acid
and rosmarinic acid. Therefore, the relationship between
antimicrobial activity and chemical composition was also
demonstrated by testing pure standards of carnosic and
rosmarinic acids. The V40 extracts with carnosic acid as the
major component of total phenolic content (Table 1)
showed activity against all bacteria tested similar to that
detected for pure carnosic acid. However, the MICs of
water-soluble (A15 and A40) extracts did not correlate well
with the results obtained for pure rosmarinic acid in all
bacteria tested. The MICs of rosmarinic acid were higher or
the same as MICs of water-soluble extracts, as determined
in S. aureus, B. cereus, and Salmonella Infantis or lower as
visible in C. jejuni culture. However, the MICs of
rosmarinic acid and water-soluble (A15 and A40) extracts
were all in the range of 1.25 to 5.0 mg/ml, determined by
the dilution method in liquid media (Table 2). In addition,
pure rosmarinic acid was much less efficient against gram-
positive bacteria and campylobacters than was carnosic
acid. Summarizing the results of antibacterial activity in
Table 2, rosemary extract formulations were less effective
against Salmonella Infantis, but they efficiently inhibited the
FIGURE 4. Bacillus cereus survival curve
during exposure to V40 and A40. Each
point represents log of the mean 6
standard deviation CFU per milliliter.
1750 KLANČNIK ET AL.
growth of gram-positive bacteria and campylobacters. The
difference in this efficiency is more obvious in the case of
V15 and V40 formulations with carnosic acid as the main
phenolic component.
Antioxidant activity. The reducing power of a
compound may serve as a significant indicator of its
potential antioxidant activity. During the reducing power
assay, the presence of reductants (antioxidants) in the tested
samples resulted in reduction of the Fe3+-ferricyanide
complex to the ferrous form (Fe2+). Fe2+ was monitored
by measurement of absorbance at 740 nm, and this linearly
increased as the concentration of rosemary extract in the
reaction mixture increased. The reducing power of V15,
V40, A15, and A40 expressed as the slope (obtained by
linear regression analysis) of the curves (not shown) is
presented in Table 1. A higher slope corresponds to a
stronger reducing power of an extract. In comparison to A15
and A40, with reducing powers of 69.5 and 124 (mg/ml)21,
respectively, V15 and V40 showed weaker reducing powers
that amounted to 27.2 and 66.1 (mg/ml)21, respectively
(Table 1).
In food and biological systems, many free radical
species are formed. The DPPH? radical has been widely
used to evaluate the free radical scavenging capacity
of antioxidants. The free radical is scavenged by an
antioxidant that donates an electron or hydrogen ion to
a radical, and therefore, a stable molecule is formed.
The effectiveness in scavenging free radicals was evaluated
as the concentration of rosemary extract in the reaction
mixture that caused a decrease in the initial DPPH?
concentration by 50% (EC50) and is presented in Table 1.
A higher EC50 value indicates a weaker capability to
scavenge DPPH? radicals. At a similar level of the main
active ingredient, the free radical scavenging effectiveness
of A15 and A40, where the EC50 was 0.0132 and
0.0074 mg/ml, respectively, was stronger than that of V15
and V40, where the EC50 amounted to 0.0227 and
0.0092 mg/ml, respectively.
Heat-induced oxidation of an aqueous emulsion system
of linoleic acid and b-carotene was used to determine the
antioxidant activity of the investigated rosemary extracts in
a heterogeneous system where the lipid and water coexisted
with an emulsifier. Peroxyl radicals formed by linoleic acid
oxidation attack the b-carotene molecule and, as a result, it
undergoes rapid decolorization. The extent of b-carotene
bleaching can be diminished or prevented by the presence of
an antioxidant that reacts with free radicals formed in the
system (42). The antioxidant activity coefficient of V15,
V40, A15, and A40 after 80 min of incubation at 50uC and
at a concentration in the emulsion of 0.04 mg/ml, calculated
according to equation 2, amounted to 0.59, 0.79, 0.12, and
0.34, respectively (Table 1).
The antioxidant activity of phenolic compounds
depends on several factors, among them the number of
hydroxyl groups and their polarity (50). Rosmarinic acid
with four hydroxyl groups attached to the phenyl ring, in
comparison to carnosic acid with two hydroxyl groups, is
more effective because more hydrogen atoms can be
J. Food Prot., Vol. 72, No. 8
donated to free radicals. Although A15 and A40 (the most
abundant phenolic compound is rosmarinic acid) showed
higher reducing powers and free radical scavenging
effectiveness than V15 and V40 did (where the most
abundant phenolic compound is carnosic acid), their
antioxidant activities in aqueous emulsion system were
weaker. These phenomena can be explained by the interfacial
properties of the constituent and their partition in the medium.
In an emulsion, the constituents more polar such as
rosmarinic acid remain in the aqueous phase, and they are
less effective in protecting linoleic acid. Constituents less
polar such as carnosic acid equilibrated into the lipid phase,
Tween micelles, and the lipid-water interfaces, and thus
exhibit a greater protective effect in prevention of oxidation
(29). These observations are in agreement with previous
results (23) showing that in emulsified corn oil, carnosic acid
was found to be one of the major antioxidant compounds of
rosemary extract that stabilize unsaturated fatty acids, and
thus retard their deterioration (22) and exhibit a greater
protective effect against oxidation than rosmarinic acid.
In conclusion, bacterial contamination and food
oxidation are the most important factors influencing food
quality loss; therefore, preventing microbial growth and
food oxidation are highly relevant to food processing. In this
study, the best antimicrobial efficiency was confirmed for
oil-soluble rosemary extract formulations against the gram-
positive bacteria B. cereus and S. aureus. Gram-negative
Salmonella exhibited marked resistance, which could be
related to the lower permeability of their surface for
phenolic compounds.
The disk diffusion method is appropriate only as a
preliminary check of antimicrobial activity prior to
quantitative determination with the dilutions methods. A
comparison of dilutions methods showed that MICs
determined with agar dilution, microdilution, and broth
macrodilution were the same in the case of Bacillus and
Staphylococcus, but taking into account all tested organ-
isms, the microdilution method with ATP measurement was
found to be a rapid and accurate way of testing antimicrobial
efficiency. Its results correlated with the MICs from survival
curves measurement.
Reducing power and free radical scavenging effective-
ness was higher in water-soluble formulations, according to
their higher total phenolic content, but in an aqueous
emulsion system of linoleic acid, they exhibited lower
antioxidant activity. Therefore, we can assume that the
phenomena at the interface between the lipid phase (lipid
bilayer of the cell membrane or droplet of emulsified linoleic
acid) and the aqueous phase are related to the partitioning
properties of these compounds determined by their lipophilic-
hydrophilic character, and are the main factors that influence
both the antimicrobial as well as the antioxidant activity in
real food systems that are heterogeneous by nature.
The present study provides additional data in support of
rosemary extracts as natural antimicrobial and antioxidant
agents. However, further research is needed to verify their
antimicrobial effectiveness in different food matrices, as
well as to evaluate their effectiveness in protecting food
quality throughout its shelf life.
J. Food Prot., Vol. 72, No. 8 ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACTS
ACKNOWLEDGMENTS
The authors thank the Ministry of Higher Education, Science, and
Technology of Republic of Slovenia for financial support of their projects.
Partial support to A.K. was also provided through Research Project
Biotracer (FP-03627).
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