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Journal of Food Protection, Vol. 72, No. 8, 2009, Pages 17441752

Copyright G, International Association for Food Protection

Research Note

In Vitro Antimicrobial and Antioxidant Activity of Commercial Rosemary Extract Formulations

NIK,1 BERNARDA GUZEJ,1 MAJDA HADOLIN KOLAR,2 HELENA ABRAMOVIC ,1 ANJA KLANC 1 SONJA SMOLE MOZINA *
1Department

AND

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 oilsoluble 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 carnosate, 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 antimicrobial 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

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TABLE 1. The contents of total phenolic compounds and of carnosic and rosmarinic acids, as well as CR, EC50, and CAA valuesa
Plant extract Total phenolic content (mg/g) Carnosic acid (mg/g) Rosmarinic acid (mg/g) CR (mg/ml)
21

EC50 (mg/ml)

CAA

V15 V40 A15 A40

a

198 438 593 915

156 6 8 407 6 5

b 161 6 4 420 6 6

27.2 66.1 69.5 124

6 6 6 6

0.4 0.6 0.3 1

0.0227 0.0092 0.0132 0.0074

6 6 6 6

0.0005 0.0002 0.0002 0.0001

0.59 0.79 0.12 0.34

6 6 6 6

0.01 0.02 0.02 0.01

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. , 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 watersoluble 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 gramnegative (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 bleaching test were selected to describe the antioxidant potential of the extracts. MATERIALS AND METHODS
Plant extracts formulations and pure substances. Commercial 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 (analytical grade), ethanol (96%), sodium carbonate (analytical grade), and trichloroacetic acid (99.5%). b-Carotene (.97%), 2,2diphenyl-2-picrylhydrazyl (DPPH?) reagent, Folin-Ciocalteu reagent, chlorogenic acid (.95%), linoleic acid (.95%), Tween 20,

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 abac, Croatia), and (analytical grade) purchased from Zorka (S 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 M9 (poultry 33560 (bovine feces isolate), and Salmonella Infantis Z 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

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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 grampositive 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 grampositive 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 corresponding 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 manufacturers 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

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 % of remainingDDPH DDPH? ~ 100 | 1 % of remaining 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 preparation (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:   Ast~0 { Ast~80 2 CAA ~ 1 { Act~0 {Act~80 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

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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 broth macrodilution tests with measurement of the kinetics of inactivation

M 9 Salmonella Infantis Z

Microdilution MICmdil

(Statgraphics Co., Tulsa, OK) was used for statistical analysis. All measurements of MICs were repeated at least in triplicate, and the most representative values were used. The results for survival curves and antioxidant measurements were independently repeated three or more times and expressed as means with standard deviation. The least-squares method was used to perform regression analysis.

Broth macrodilution

MBC

20.0 20.0 20.0 20.0 10.0 5.0 5.0 2.5 2.5 5.0 5.0 5.0 2.5 2.5 5.0 .100.0 100.0 100.0 100.0 ND 2.5 0.313 5.0 5.0 0.313 1.25 0.156 2.5 2.5 0.156 1.25 0.156 2.5 2.5 0.156 40.0 20.0 .40.0 .40.0 ND 0.156 0.156 5.0 5.0 0.156 0.156 0.156 5.0 5.0 0.156 0.156 0.156 5.0 5.0 0.156 0.625 0.156 5.0 5.0 0.156 5.0 0.625 20.0 20.0 ND a 0.156 0.078 2.5 1.25 0.078 0.156 0.078 2.5 1.25 0.078 0.156 0.078 5.0 1.25 0.156 8.0 7.0 10.0 5.0 5.0 9.0 9.0 9.0 8.0 5.0

MIC

RESULTS AND DISCUSSION Antimicrobial activity. The effect of rosemary extracts was examined against the foodborne pathogenic bacteria S. aureus, B. cereus, C. jejuni, and Salmonella Infantis. Grampositive strains were much more sensitive to rosemary extracts. For example, by the agar dilution method, the MICdil for S. aureus and B. cereus was 0.078 to 5.0 mg/ml, whereas for Salmonella, it was 5.0 to 10.0 mg/ml (Table 2). Campylobacter seemed to be quite sensitive to the various extracts tested, which is consistent with earlier reports (18, 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 susceptible than was S. aureus. The gram-negative Salmonella exhibited marked resistance to the extract formulations tested. The outer membrane surrounding the cell wall in gram-negative bacteria, which restricts diffusion of compounds through its lipopolysaccharide covering, may be the reason for this resistance (19, 55). The single membrane of gram-positive bacteria is considerably more susceptible to permeation by antimicrobials. The sensitivity of bacteria to polyphenols also depends on the bacterial species and polyphenol structure (44, 53, 60). This was significant for the bacterium Campylobacter, which has survival strategies different from other foodborne bacteria, because it lacks stress adaptive responses described in other enteropathogens (36, 38, 39). For preliminary screening of antibacterial activity, the disk diffusion method was used. However, some extracts showed very low efficiency. Water-soluble (A15 and A40) formulations gave a minimal inhibition zone against gramnegative C. jejuni and Salmonella Infantis only at concentrations of 40.0 and 100.0 mg/ml, respectively (Table 2). As reported by Moreno et al. (34), the absence of an inhibition zone does not necessarily mean an inactive compound. Compounds less polar diffuse more slowly into the culture medium. The diameter of the inhibition zone is influenced by the rate of diffusion of the antimicrobial agent through the agar, and the hydrophobic nature of most plant extracts prevents uniform diffusion of these substances through agar media (20, 47). Thus, a quantitative determination of antibacterial activity was made through the agar dilution method, where activity was shown at lower concentrations compared with the disk diffusion method (Table 2). Oil-soluble formulations (V15 and V40) inhibited growth of B. cereus and S. aureus at 0.078 to 0.625 mg/ml, while water-soluble formulations (A15 and A40) required 1.25 to 5.0 mg/ml. Since the agar dilution method is time- and materialconsuming, we introduced the broth microdilution method as a rapid quantitative determination of MIC based on measurement of the bioluminescent signal, which is

Agar Disk diffusion dilution MICdil MICdif

Broth macrodilution

MBC

Campylobacter jejuni ATCC 33560

MIC

Microdilution MICmdil

Agar Disk diffusion dilution MICdil MICdif

Broth macrodilution

MBC

Staphylococcus aureus ATCC 25923

MIC

Microdilution MICmdil

Agar Disk diffusion dilution MBC MICdif MICdil

Broth macrodilution

Bacillus cereus WSBC 10530

MIC

Microdilution MICmdil

Agar Disk diffusion dilution MIC (mg/ml) MICdif MICdil

V15 0.625 V40 0.313 A15 20.0 A40 5.0 Carnosic acid NDb Rosmarinic acid ND

6.0
a b

5.0

5.0

ND

10.0

5.0

2.5

5.0

ND

1.25

1.25

1.25

2.5

ND

1.25

2.5

2.5

5.0

, not achieved. ND, not defined.

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FIGURE 1. Staphylococcus aureus survival 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 concentrations found to be inhibitory by the diffusion and dilution tests (Figs. 1 through 4). In some cases, higher concentrations 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.

effect on C. jejuni cells in liquid media by the broth macrodilution and microdilution methods, probably indicating 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 destroyed 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

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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.

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. Vegetative 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 measurement 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

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 FolinCiocalteu 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 grampositive 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.

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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

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 grampositive 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 organisms, 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 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. 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 lipophilichydrophilic 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.

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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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