Effect of Zataria multiflora Boiss. Essential Oil, Nisin,and Their Combination on the Production of Enterotoxin C

and a-Hemolysin by Staphylococcus aureus

Mahnoosh Parsaeimehr,1,2 Afshin Akhondzadeh Basti,1 Behrad Radmehr,3 Ali Misaghi,1

Arash Abbasifar,1 Ghity Karim,1 Nourdahr Rokni,1 Mohammad Sobhani Motlagh,4

Hassan Gandomi,1 Negin Noori,1 and Ali Khanjari1

Abstract

The effect of different concentrations of Zataria multiflora Boiss. essential oil (EO; 0%, 0.005%, and 0.015%), nisin(0, 0.125, and 0.25 mL=mL), and their combinations on the production of staphylococcal enterotoxin C (SEC) anda-hemolysin (a-toxin) by Staphylococcus aureus at different inoculation levels (103, 104, and 105 cfu=mL) in brainheart infusion broth during storage at 358C for up to 43 days was evaluated. The SEC production was signifi-cantly ( p< 0.05) inhibited and the hemolysis due to a-toxin was significantly ( p< 0.05) reduced by EO con-centration at levels 0.015% and 0.005%, respectively. Significant ( p< 0.05) inhibitory effect of EO on SECproduction at level 0.005% was observed when it was used in combination with nisin¼ 0.125 mL=mL. Thesignificant ( p< 0.05) synergistic effect of EO¼ 0.005% and nisin¼ 0.125 mL=mL was also observed as morereduction of hemolysis due to a-toxin than EO¼ 0.005% alone. Further, EO significantly ( p< 0.05) preventedSEC production by S. aureus during the manufacturing process of a traditional Iranian white brined cheese (as afood model) even at its lowest concentration (5 mL=100 mL), in this study.

Introduction

Staphylococcus aureus is a pathogen of major concernfor clinical infection and foodborne illness. S. aureus pro-

duces a number of extracellular proteins, for example,staphylococcal enterotoxins (SEs) and hemolysins (a, b, g, andd) which may have a role in the pathogenicity of staphylo-coccal diseases (Palmer et al., 2004; Chiang et al., 2008).

Concerns over the safety of some chemical preservativesand negative consumer reactions to preservatives perceivedas chemical and artificial have prompted on increased interestin more ‘‘natural-green’’ alternatives for the maintenance orextension of product shelf-life (Tassou et al., 2000; Palmer et al.,2001). However, to establish the usefulness of natural anti-microbial preservatives, they must be evaluated alone and incombination with other preservation factors to determinewhether there are synergistic effects and to devise multiplehurdles (Lopez-Malo et al., 1998; Pol and Smid, 1999; Pol et al.,2002).

Zataria multiflora Boiss. is a plant belonging to the Lami-naceae family, which geographically grows only in Iran,

Pakistan, and Afghanistan. This plant is extensively used as aflavor ingredient in a wide variety of foods in Iran. Themain constituents of the essential oil (EO) of this plant arephenolic compounds such as carvacrol and thymol (Basti et al.,2007).

In our previous works, Basti et al. (2007) and Moosavy et al.(2008) reported that Z. multiflora Boiss. had inhibitory effect(growth inhibition) on Salmonella Typhimurium and S. aureusin brain heart infusion (BHI) broth and in a food modelsystem.

In this study, the effect of different concentrations ofZ. multiflora Boiss. EO, nisin, and their combinations on en-terotoxin C and a-hemolysin (a-toxin) production by S. aureusin BHI broth was evaluated.

In addition, we studied the effect of EO on the entero-toxigenic ability of S. aureus during the manufacturing processof white brined cheese (traditional Iranian white brinedcheese as a food model), which has been involved in theS. aureus food-poisoning outbreaks; S. aureus is considered asa treat to the safety of cheese (Stecchini et al., 1991; Luca et al.,1997; Buyser et al., 2001; Carmo et al., 2002; Arques et al., 2005;

1Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.2Faculty of Agriculture, Islamic Azad University—Shushtar Branch, Tehran, Iran.3Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Islamic Azad University—Karaj Branch, Tehran, Iran.4Iranian Veterinary Organization, Tehran, Iran.

FOODBORNE PATHOGENS AND DISEASEVolume 7, Number 3, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=fpd.2009.0416

299

Kaan Tekinsen and Ozdemir, 2006; Kerouanton et al., 2007;Akineden et al., 2008).

Materials and Methods

Plant material

Z. multiflora Boiss. was collected in the Shiraz province ofIran and identified by the Institute of Medicinal Plants,Medical University of Tehran, Iran. The method of extractionand percentages of components of the EO (as determined bygas chromatography [GC]–mass spectrometry [MS]) havebeen described by Basti et al. (2007).

Nisin

Nisin containing 2.5% active nisin was purchased fromSigma-Aldrich (Irvine, Ayrshire, United Kingdom; EC 215-807-5). Nisin stock solution was prepared with 0.02 M HCl(pH 1.6) and was filter sterilized through a 0.45-mm sterile,disposable, and nonpyrogenic syringe filter (Nalgene, Ro-chester, NY).

Preparation of inoculum

The inoculum of S. aureus ATCC 6538 was prepared bytransferring cells from working culture to a tube of BHI broth.After 18 h of incubation at 358C, the second subculture wasprepared and incubated for 18 h at 358C. In a 13�100 mmsterile cuvette, the S. aureus broth culture was adjusted to anoptical density (OD) (absorbance) of 0.02 at 600 nm, using aSpectronic 20 spectrophotometer (Milton Roy Company, Ivy-land, PA). This adjustment gave a cell concentration of 1�107

cfu=mL for Staphylococcus as determined in a preliminary trial.The number of cells in the suspension was determined byduplicate plating from 10-fold serial dilutions on BHI agar andcounting the colonies after 24-h incubation at 358C.

Experimental design to assess the effect of EO, nisin,and their combination on staphylococcal enterotoxin Cand a-toxin production by S. aureus in BHI broth

To assess the effect of different concentrations of EO, nisin,and their combinations on the production of staphylococcalenterotoxin C (SEC) and a-toxin by S. aureus, the experimentwas arranged in a factorial design. This design (3�3) includedthree levels of EO (0%, 0.005%, and 0.015%), three levels ofnisin (0, 0.15, and 0.25 mL=mL), repeated observations (18times) for visible growth (turbidity) in BHI broth, and sub-sequent analysis for SEC detection and a-toxin activity for upto 43 days.

Preparation of broth substrate. BHI powder (3.7 g) wasdissolved in 90 mL distilled water in a 250-mL screw-cappedflask by mild heating. Then, EO was added in differentamounts (0%, 0.005%, and 0.015%). To produce and maintaina stable oil–water emulsion in broth substrate during the pe-riod of study (43 days), we used the method explained byMann and Markham (1998) with some modifications. Briefly,we added 5% (v=v) dimethylsulfoxide (Merck SchunchardtOHG, Hohenbrunn, Germany) as an emulsifier and 0.05%(w=v) agar-agar (Merck KGaA, Darmstadt, Germany) as astabilizer to the broth substrate. The same amounts of di-methylsulfoxide and agar-agar were also added to the com-

bination with no EO (0%) and nisin (0 mL=mL), consideringany likely effects of them on growth and toxin production ofthe test organism. The final volume of broth substrate wasbrought to 100 mL with additional distilled water. The contentof each flask was autoclaved at 1218C for 15 min. After cool-ing, aliquots of the appropriate concentration of the nisin(stock solution) were added to the tubes so as to give finalconcentrations of 0.0, 0.015, and 0.25 mL=mL (nisin concen-trations in the present report are expressed as active nisinconcentrations). The content of each flask was dispensed intosterilized screw-capped tubes at levels of 9 mL per tube.

Inoculation and storage of broth substrates. Tenfold se-rial dilutions of S. aureus culture (OD600nm¼ 0.02) were pre-pared, using a set of seven tubes (20 mL size), each tubecontaining 9 mL BHI broth with a designated combination ofEO-nisin. The last six tubes used for this study gave a range ofinoculum levels from 105 to 100 organisms=mL. The inoculatedtubes were stored at 358C for up to 43 days. During theseperiods all the tubes were observed for visible turbidity (andconsequently were confirmed by plate count to have>107 or-ganisms=mL) at 18 time intervals (1, 2, 3, 4, 5, 6, 7, 8, 13, 16, 19,22, 25, 28, 31, 34, 37, and 43 days). The tubes showing growth(visible turbidity) at a particular observation during the studywere analyzed for SEC detection and a-toxin activity.

Enterotoxin detection. The RIDASCREEN SET kit(R-Biopharm, Darmstadt, Germany), a commercial SE visualimmunoassay kit with a minimum detectable limit of 0.50 to0.75 ng of SEs per mL or g of sample, was used for SE detec-tion in the positive tubes (tubes showing visible growth at aparticular observation during the study). The kit utilizesmonovalent capture antibodies against SE types A to E;therefore, it simultaneously detects and identifies the entero-toxin types. Enterotoxin assay was performed using themethod recommended by the manufacturer of the kit (Parket al., 1994). The OD of colored extracts resulting from theenzymic reactions of the RIDASCREEN kit was determined at450 nm using a microplate reader (StatFax 2100; AwarenessTechnology Inc., Palm City, FL).

Determination of a-toxin activity. The a-toxin activity wasmeasured according to the method described by Palmer et al.(2004). Supernatants from culture of positive tubes were ob-tained by centrifugation of the cultures at 1600g for 10 min at48C (centrifuge 5810R; Eppendorf, Hamburg, Germany). Thenrabbit erythrocytes (Department of Microbiology, Faculty ofVeterinary Medicine, University of Tehran) were added to1 mL sample of each culture supernatant with 450mL ofphosphate-buffered saline (PBS; pH 7.2) to achieve a finalconcentration of 1% in a microcentrifuge tube. The tubes weregently mixed by inversion, incubated at 378C for 30 min, andthen centrifuged at 1000 g for 5 min at 48C (centrifuge 5810R;Eppendorf ). The A630 was read using enzyme-linked immu-nosorbent assay reader (StatFax 2100; Awareness TechnologyInc.). To determine the percentage of hemolysis, controls wereset up, which contained 1% erythrocytes and deionized water.The results were expressed as the percentage of hemolysiscompared with water-lysed control, which was taken as 100%hemolysis. No significant hemolysis was detected in otherprepared controls containing PBS (pH 7.2), BHI broth, or BHIbroth with different combinations of EO and nisin.

300 PARSAEIMEHR ET AL.

Determination of hemolysis due to intracellular a-toxinactivity after culture with EO, nisin, and their combina-tion. Experiment was conducted to determine the likely ef-fect of different concentrations of EO, nisin, and theircombinations on S. aureus cells’ inability to export a-toxin andsubsequent intracellular accumulation of the toxin using themethod described by Palmer et al. (2004). The supernatantobtained from the culture of each positive tube was discarded.The cells were washed twice in PBS (pH 7.2) and resuspendedin 10 mL PBS (pH 7.2). Cells were lysed on ice by sonication(UP 200S Ultraschall processor; Dr. Hielscher GmbH, Stutt-gart, Germany) for 5 min and examined by light microscopyto ensure of total lysis of the cells. To remove the cell debris,the lysate was centrifuged at 1600 g for 10 min at 48C (cen-trifuge 5810R; Eppendorf ) and the hemolysis due to intra-cellular a-toxin activity was determined in the supernatant asearlier (Palmer et al., 2004).

Determination of direct effect of EO, nisin, and theircombination on a-toxin activity. Different designatedamounts of EO (0%, 0.005%, and 0.015%), nisin (0, 0.15, and0.25 mL=mL), and their combinations were added to tubescontaining 10 mL supernatant of BHI broth which was cul-tured with S. aureus alone and showed visible turbidity(therefore known to have a-toxin). All tubes were incubated at378C for 1 h with shaking. Then, the samples were left to settlefor 15 min (Palmer et al., 2004). One milliliter of sample fromeach tube was taken and the activity of a-toxin was measuredas earlier (Palmer et al., 2004).

Experimental design to assess the effectof EO on SE production by S. aureusduring the manufacturing processof a traditional Iranian white brined cheese

This design included three levels of EO (0, 5, and15 mL=100 mL or 0%, 0.005%, and 0.015%), repeated exami-nations (eight times) for growth, and subsequent analysis forSE detection during the manufacturing process of a traditionalIranian white brined cheese for up to 60 days.

Manufacturing and sampling of a traditional Iranian whitebrined cheese. Traditional Iranian white brined cheese wasmanufactured according to the procedure outlined here:pasteurized (75.08C=16 s) cow’s milk (with a fat content of2.5%) was placed in a small stainless steel cheese vat rest-ing on a larger pilot-plant–sized, steam-jacketed cheese vatpartially filled with water. Then different concentrations ofEO were added according to designated groups in thisstudy. After warming the milk up to 358C, it was inoculatedwith the test organisms at 1�103 cfu=mL in separatedgroups. Then 2 mL of Dutch State Mines (DSM)-Microbialrennet, diluted 1:40 in sterile distilled water, was added alongwith 0.2 mg=mL calcium chloride. The coagulum was cut witha sterile 7.5-cm knife 1 h after the addition of the rennet. Thenthe cut curd was transferred into rectangular (28�12�12 cm3)metal hoops and allowed to drain for 6 h. After draining atroom temperature (228C), the cheese was divided into pieces(12�8�6 cm3) and placed into 20% sterile salt brine for 8 h at228C. Then, pieces of the cheese were removed from the brine,placed into a sterile 500-mL capacity glass container, andcovered with 8% sterile salt brine. Cheese in this brine was

ripened at 148C for 15 days, and then cheese in the brine wasmoved to storage at 48C.

Samples for S. aureus enumeration and detection of SEswere taken during manufacturing, ripening, and storage ofthe white brined cheese according to the following schedule:(a) pasteurized milk, (b) inoculated milk, (c) curd after cutting,(d) whey after draining of the curd, and (f ) curd after 6 h ofdraining. Also, samples were taken from the cheese at days 7,15, 30, 45, and 60. The brine used during ripening and storageof this kind of cheese was tested for S. aureus enumeration anddetection of SEs, as well.

The concentrations of EO in the final products were ob-tained using GC apparatus (Detector FID, Column HP5; GCYounglin Instrument Co. Ltd., Anyang, South Korea) usinghelium and hydrogen combined with the air as the carrier andfuel gases, respectively. Before this operation, to samplepreparation, 30 g of every sample of cheese was mincedcompletely, dissolved with 5 mL of n-hexane followed by10 mL of distilled water, and then centrifuged at 10,000 g(Avanti Centrifuge, J-25 I; GMI, Inc., Ramsey, MN) for 5 min.After removing the supernatant, the extract was filtered usinga 0.2-mm Wattman filter paper and injected into the GCapparatus.

Table 1. The Effect of Different Concentrations

of Essential Oil, Nisin, and Their Combinations

on Enterotoxin C Production by Staphylococcus

aureus at Different Inoculation Levels in Brain

Heart Infusion Broth Which Showed Visual

Turbidity During 43 Days of Storage at 358C

EO(%)

N(mL=mL)

Inoculation level(cfu=mL) Enterotoxin

0 0 103 þ0 0 104 þ0 0 105 þ0 0.125 103 þ0 0.125 104 þ0 0.125 105 þ0 0.25 103 þ0 0.25 104 þ0 0.25 105 þ0.005 0 103 þ0.005 0 104 þ0.005 0 105 þ0.015 0 103 �0.015 0 104 �0.015 0 105 �0.005 0.125 103 �0.005 0.125 104 �0.005 0.125 105 �0.015 0.125 103 �0.015 0.125 104 �0.015 0.125 105 �0.005 0.25 103 �0.005 0.25 104 �0.005 0.25 105 �0.015 0.25 103 �0.015 0.25 104 �0.015 0.25 105 �

EO, essential oil; N, nisin.

EFFECT OF ZATARIA MULTIFLORA ON TOXINS OF S. AUREUS 301

Bacterial enumeration. S. aureus count was done accord-ing to the method described by APHA (2001) using Baird-Parker agar (Merck, KGaA, Darmstadt, Germany).

Enterotoxin detection. The same RIDASCREEN SET kit(R-Biopharm) was used to evaluate the samples. Only thosesamples that showed S. aureus count upper than the minimumdetection limit for the enzyme-linked immunosorbent assaymethod (5.5�105 cfu=g) (Park et al., 1994) were analyzed forSE detection.

All experiments were conducted in triplicate. The mean ofmeasurements and corresponding standard error of the meansare given in the Results section. The main and interactive ef-fect of EO and nisin concentrations on enterotoxin productionand hemolysis due to a-toxin were evaluated by chi-square testand analysis of variance, respectively, using SPSS 15.0 statis-tical software (SPSS 15.0 for windows; SPSS Inc., Chicago, IL).Results were considered statistically significant when p� 0.05.

Sensory evaluation

Sensory-derived effects of adding different amounts ofZ. multiflora Boiss. EO (0, 5, 15, and 30mL=100 mL) to whitebrined cheese (during manufacturing process) were evaluatedby an acceptance test. The white brined cheese with different

amounts of EO was equally divided into seven parts (each partabout 500 g) and placed on white plates. The sensory evalua-tion was performed by a panel of seven judges consisting ofthe scientific staff of the Department of Food Hygiene, Facultyof Veterinary Medicine, University of Tehran, chosen for theirexperience in the sensory analysis of food. Each panelistevaluated the samples by rating, using a 9-point scale, where9¼ like extremely, 8¼ like very much, 7¼ like moderately,6¼ like slightly, 5¼neither like nor dislike, 4¼dislike slightly,3¼dislike moderately, 2¼dislike very much, and 1¼dislikeextremely, for various characteristics such as color (appear-ance), odor, and flavor. The variability of acceptance or likingof the samples was analyzed by analysis of variance andFisher’s least significant difference procedure, using SPSS 15.0statistical software (SPSS 15.0 for Windows; SPSS).

Results

Chemical composition of Z. multiflora Boiss. EO

GC-MS analysis resulted in the identification of 12 com-ponents representing 91.9% of the oil. The major compoundwas phenolic monoterpene carvacrol (71.1%); other importantcompounds were gammaterpinene (7.34%), alpha-pinene(4.26%), and eucalyptol (3.37%).

Table 2. Percentage of Hemolysis (Mean� Standard Deviation) Due to a-Toxin Produced by Staphylococcus

aureus (Cultured in Brain Heart Infusion Broth with Different Concentrations of Essential Oil, Nisin,

and Their Combinations, or in Brain Heart Infusion Broth Alone After Direct Effect of Designated Factors)

and Due to Intracellular a-Toxin Activity After Culture with Designated Factors

EO(%)

N(mL=mL)

Inoculationlevel

(cfu=mL)

Hemolysis (%) in thesupernatant of the cultureof S. aureus in BHI broth

with designated factors

Hemolysis (%) in thesupernant of the culture of

S. aureus in BHI broth alone,after addition of designated factors

Hemolysis (%) dueto intracellular a-toxinactivity after culture

with designated factors

103 0 0 42.6� 1.3 41.9� 1.19 9.4� 0.9104 0 0 43.7� 0.2 42.7� 1.1 10.3� 1.2105 0 0 43.4� 0.6 42.4� 1.3 10.1� 2.4103 0.125 0 21.1� 0.6 44� 0.2 9.6� 0.4104 0.125 0 20.8� 0.7 44.4� 0.2 9.9� 1.1105 0.125 0 20.5� 0.2 44� 0.8 9.1� 0.4103 0.25 0 20.8� 01 40.4� 0.8 8.7� 0.4104 0.25 0 19.6� 0.3 38.3� 0.2 7.7� 0.4105 0.25 0 21.2� 0.6 39.3� 0.2 7.1� 0.2103 0 0.005 41� 1.6 40� 1.3 9.6� 0.6104 0 0.005 41.4� 2.1 40� 1.3 10� 3105 0 0.005 41.1� 2.8 40.6� 2.5 7.9� 1.4103 0 0.015 41� 0.3 39.1� 0.3 6.4� 0.4104 0 0.015 41.4� 0.6 39.2� 0.3 10.4� 1105 0 0.015 41� 1.6 43� 0.2 10.9� 1.1103 0.125 0.005 13.1� 0 43.8� .5 7.8� 1104 0.125 0.005 13.5� 2.6 41.9� 0.4 9.4� 0.3105 0.125 0.005 13.4� 1.9 42.3� 0.27 8.6� 1103 0.125 0.015 12.1� 2.7 41.5� 0.3 9.5� 1104 0.125 0.015 14.3� 0.5 46.6� 0.5 8.7� 2.2105 0.125 0.015 9.1� 0.5 46.3� 0.1 8.1� 1.4103 0.25 0.005 10.7� 0.7 42.9� 1.6 9.7� 0.6104 0.25 0.005 11.2� 1.1 41.8� 2.3 8.4� 2.1105 0.25 0.005 10.9� 1.1 42.8� 2.2 8.9� 0.4103 0.25 0.015 10.2� 0.9 41.5� 2.4 8� 1.3104 0.25 0.015 10.5� 1.7 42.6� 1 7.7� 0.6105 0.25 0.015 10.7� 1.9 41.8� 1.4 8.1� 1.8

BHI, brain heart infusion.

302 PARSAEIMEHR ET AL.

Enterotoxin analysis (for BHI broth)

Only the first three inoculum levels (105, 104, and 103

organisms=mL) out of six which finally showed visibleturbidity at certain intervals (from 18 time intervals) duringthe study (data not shown) were analyzed for enterotoxindetection (Table 1). The organism used in this study (S. aureusATCC 6538) produced only enterotoxin C in the control group(with no EO and nisin) in BHI broth.

As the results show, EO at level 0.005% had no inhibitoryeffect on enterotoxin C production by S. aureus in this study.By increasing the concentration of EO to 0.015%, significant( p< 0.05) inhibitory effect on enterotoxin C production wasobserved. Significant ( p< 0.05) inhibitory effect of EO on SECproduction at a level of 0.005% was observed when it wasused in combination with nisin¼ 0.15 mL=mL.

Different concentrations of nisin had no inhibitory effect onenterotoxin production when used alone in this study. Also,no significant ( p> 0.05) difference was observed between theeffect of different inoculation levels of S. aureus on toxinproduction in the same combination.

Hemolysis due to a-toxin activity

According to the results (Table 2), 42.59–43.70% hemolysisdue to a-toxin activity of S. aureus was obtained in combina-tion with EO¼ 0% and nisin¼ 0mL=mL (control groups). Thiswas significantly ( p< 0.05) reduced (17.43–20.53%) by EOconcentration at a level of 0.005%. The significant ( p< 0.05)synergistic effect of EO¼ 0.005% and n¼ 0.125mL=mL wasalso observed as reduction of a-toxin activity, and the per-centage of hemolysis reached to 13.14–13.52%.

Similar to the results which were obtained for enterotoxinproduction, different concentrations of nisin had no signifi-cant ( p> 0.05) effect on a-toxin activity when used alone, andno significant ( p> 0.05) difference was observed between theeffect of different inoculation levels on a-toxin production inthe same combination in this study.

Table 2 indicates that various amounts of EO, nisin, andtheir combinations had no significant ( p> 0.05) direct effecton a-toxin activity when added to the supernatant of BHIbroth (which was cultured with S. aureus alone). Therefore, allof the combinations showed hemolysis values 38.25–46.58%.

When the cells of tubes (containing BHI broth with differ-ent concentrations of EO, nisin, and their combinations cul-tured with S. aureus) were lysed, little hemolytic activity wasobtained (Table 2).

Enterotoxin analysis (for food system)

Table 3 represents the growth response (log10 cfu=mL or g)of S. aureus as affected by EO during 60 days of manufacture,ripening, and storage of traditional Iranian white brinedcheese and shows the results of SE detection which was as-sessed only in samples that showed an S. aureus count of morethan 5.5�105 cfu=g.

S. aureus was not isolated in pasteurized milk before inoc-ulation. Also, SEs were not detected in pasteurized milk beforeand just after inoculation of S. aureus. As the results show, noSEs were detected in the cheeses except for the control groupwithout EO having more than 1�107 cfu=g of S. aureus afterthe first 15 days of aging which were positive only for SEC.The SEs were also not detected in the studied brine.

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EFFECT OF ZATARIA MULTIFLORA ON TOXINS OF S. AUREUS 303

EO significantly ( p< 0.05) prevent the SEC production byS. aureus during manufacturing process of traditional Iranianwhite brined cheese even at its lowest studied concentration(5 mL=100 mL), in this study.

The concentrations of EO in the final aged cheeses weredifferent from the primary injected levels in basic milks. Thevalues (mean� standard deviation) were 8.33� 0.57,16.02� 1.52, and 28.05� 1.43 mL=100 g for injected milks atconcentration levels 5, 15, and 30 mL=100 mL, respectively.Some parts of EO phenolic compounds were released intothe aqueous phase, because these substances are polar tosome extent and are soluble partially in polar solvents such aswater.

Organoleptic effects of Z. multiflora Boiss. EO

Table 4 shows the mean acceptability scores of the whitebrined cheese samples containing EO. Indeed the samplescontaining 5, 15, and 30 mL=100 mL of the EO were acceptableto panelists. However, the most preferred sample containedEO 5 mL=100 mL.

Discussion

In our previous works, we found a significant inhibitoryeffect of Z. multiflora Boiss. EO on the growth of S. aureus inBHI broth (Basti et al., 2007) and in a food model system(Moosavy et al., 2008).

The results obtained in this study showed the potentialability of Z. multiflora Boiss. EO to significantly inhibit andreduce enterotoxin C and a-toxin production, respectively.The ability of Z. multiflora Boiss. EO to inhibit enterotoxinproduction of S. aureus in BHI broth and in a traditionalIranian white brined cheese (even at its lowest studied con-centration) in this study indicates its potential as a novel‘‘natural-green’’ food preservative. Similar findings were ob-served by Palmer et al. (2004), which showed the significantability of subinhibitory concentrations of bay, cinnamon, andclove EOs to decrease the production of enterotoxin A anda-toxin of S. aureus in tripton soya broth.

As the results show (Table 3), the SEs were not detectedin the control group (cheese with EO¼ 0mL=100 g) having anS. aureus count equal to 5.86 log10 cfu=g after 7.5 h of proces-sing. This was similar to the findings of Fujikawa and Mor-ozumi (2006), who have indicated the necessity of S. aureuscell concentration of about 6.5 log10 cfu=mL in milk in order toincrease SEA linearly with time which can be detectable.

Despite the strong antimicrobial activity against foodbornepathogens and spoilage microorganisms shown by EOs (Tas-

sou and Nychas, 1995; Tassou et al., 2000; Rhayour et al., 2003;Bagamboula et al., 2004; Oussalah et al., 2006; Basti et al., 2007),their practical application is currently limited because of theundesirable flavor changes they cause in food products (Pal-mer et al., 2001; Yamazaki et al., 2004). Leistner and Gorris(1995) suggested that food preservation by multiple preser-vatives in small amounts was superior to preservation by alarge amount of a single preservative for both secure microbialstability and safety and for maintaining the sensory, nutritive,and economic properties of foods. The synergistic effect be-tween EO’s constituents and other antimicrobial substancessuch as nisin has been conclusively demonstrated (Pol andSmid, 1999; Ettayebi et al., 2000; Periago and Moezelaar, 2001;Pol et al., 2002) and it has been noted that the activities of EOand its constituents are enhanced by the presence of nisin(Yamazaki et al., 2004; Rajkovic et al., 2005; Misaghi and Basti,2007; Moosavy et al., 2008). In this study, we also found thesignificant synergistic effect of Z. multiflora Boiss. EO and nisinon enterotoxin and a-toxin production by S. aureus.

According to the results (Table 2), it seems unlikely that EO,nisin, and their combination were able to inhibit the activity ofa-toxin directly and had effect on accumulation of intracel-lular a-toxin because of cells’ inability to successfully exportthe toxin. These were similar to the findings of Palmer et al.(2004), who showed that the subinhibitory concentrations ofbay, cinnamon, and clove EOs had no direct effect on a-toxinactivity when added to the supernatant of tripton soya brothcultured with S. aureus alone. Similar to our results, they didnot find any effect of the studied EOs on the accumulation ofintracellular a-toxin.

Conclusion

The results indicated the potential effect of Z. multifloraBoiss. EO to inhibit enterotoxin production and reduction ofa-toxin activity of S. aureus.

The significant ( p< 0.05) synergistic effect of Z. multifloraBoiss. EO and nisin on the inhibition of enterotoxin produc-tion and reduction of a-toxin activity of S. aureus, which wasshown in this study, can improve the scope of EO function inthe food industry.

Acknowledgment

The authors acknowledge the financial support from theAzad University-Shushtar branch and the Faculty of Veter-inary Medicine, University of Tehran, for this research.

Disclosure Statement

No competing financial interests exist.

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Table 4. Mean Rating for the Acceptability

of White Brined Cheese Containing Various

Concentrations of Zataria multiflora Boiss.

Essential Oil

EO (mL=100 mL) Mean rating� SD

0 8.00� 0.00a

5 8.14� 0.40a

15 7.85� 1.46a

30 6.24� 1.51b

Means followed by the same superscript letters are not signifi-cantly different ( p< 0.05).

304 PARSAEIMEHR ET AL.

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Address correspondence to:Afshin Akhondzadeh Basti, D.V.M., Ph.D.

Department of Food HygieneFaculty of Veterinary Medicine

University of Tehran14155-6453 Tehran

Iran

E-mail: aakhond@ut.ac.ir

EFFECT OF ZATARIA MULTIFLORA ON TOXINS OF S. AUREUS 305