Bulgarian Journal of Veterinary Medicine, 2018 ONLINE FIRST ISSN ; DOI: /bjvm.2136

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1 Bulgrin Journl of Veterinry Mediine, 2018 ONLINE FIRST ISSN ; DOI: /jvm.2136 Originl rtile ANTIBACTERIAL EFFECTS OF ZIZIPHORA CLINOPODIOIDES AND MENTHA SPICATA ESSENTIAL OILS AGAINST COMMON FOOD-BORNE PATHOGEN BIOFILMS ON STAINLESS STEEL SURFACE Summry Y. SHAHBAZI Deprtment of Food Hygiene nd Qulity Control, Fulty of Veterinry Mediine, Rzi University, Kermnshh, Irn Shhzi, Y., Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon food-orne pthogen iofilms on stinless steel surfe. Bulg. J. Vet. Med. (online first). The im of the urrent study ws to ssess the effets of Ziziphor linopodioides independently nd in omintion with Menth spit essentil oils ginst iofilms of six food-orne pthogens inluding Listeri monoytogenes, Slmonell Typhimurium, Esherihi oli O157:H7, Billus sutilis, Billus ereus nd Stphyloous ureus developed on the surfe of stinless steel oupons. The minimum inhiitory onentrtions (MICs) of essentil oils (EOs) were determined using roth miro-dilution method. The order of dhered ell ounts in iofilms on the stinless steel surfes ws s followed: S. Typhimurium (5.75 log CFU/m 2 ) > E. oli O157:H7 (5.31 log CFU/m 2 ) > S. ureus (5.21 log CFU/m 2 ) > L. monoytogenes (4.62 log CFU/m 2 ) > B. sutilis (4.23 log CFU/m 2 ) > B. ereus (4.17 log CFU/m 2 ). There ws signifint differene etween the popultion of ll investigted pthogens on the stinless steel oupons treted with ZEO nd MEO (P<0.05). The numer of surfe-dhered ells signifintly ws deresed fter 1.5, 3 nd 4.5 min of exposure to the disinfetnt solutions sed on ZEO seprtely nd in omintion with MEO (P<0.05). In ll teril iofilms treted with the omintion of ZEO nd MEO logrithmi redutions higher thn 2 log CFU/m 2 were oserved. The highest iofilm removl tivity of disinfetnt solutions sed on ZEO nd MEO ws found for B. ereus, followed y B. sutilis, L. monoytogenes, S. ureus, E. oli O157:H7 nd S. Typhimurium, respetively. It n e onluded tht the disinfetnt solution sed on the omintion of ZEO nd MEO n e used s n pproprite nturl disinfetnt to ontrol iofilm formtion in food proessing filities nd utensils. Key words: iofilm, Menth spit, stinless steel surfe, Ziziphor linopodioides INTRODUCTION The dhesion of teril pthogens to surfes of food hndling, storge nd proessing equipment leds to rossontmintion of foods, whih n e re-

2 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. sponsile of food-orne diseses nd importnt eonomi losses (Be et l., 2012; Kud et l., 2016). Stinless steel is the most preferred mteril for friting food proessing filities nd utensils suh s ontiners, pipelines nd working surfes due to its high mehnil strength, resistne to orrosion nd longevity (Kusumningrum et l., 2003; Csrin et l., 2014). Previous studies hve shown tht Listeri monoytogenes, Slmonell Typhimurium, Esherihi oli O157:H7, Billus sutilis, Billus ereus nd Stphyloous ureus re ple of dhering nd form iofilms on stinless steel surfes (Shlisselerg & Yron, 2013; Ouli et l., 2014; Csrin et l., 2014; Kim et l., 2016; Kud et l., 2016). It hs een suggested tht ommonly used disinfetnts in food proessing environments inluding sodium hypohlorite, sodium hydroxide nd enzlkonium hloride re ple of erditing iofilms developed y forementioned pthogens on stinless steel surfe to vrying extent (Nm et l., 2014; Chen et l., 2015). Therefore, further investigtions for development of onepts nd the disovery of novel strong ntimiroil ompounds to derese the risk of ontmintion in food setor is of unquestionle interest. On the other hnd, due to reent inresing onsumer onerns out the toxi residues of lssil syntheti snitisers these reserh efforts must e shifted on nturl lterntives (Ryu et l., 2004; Rffell et l., 2017). Relted to this topi, plnt essentil oils (EOs) nd extrts hve ttrted reserh interest to ontrol teril iofilms in food industries (Bzrgni & Rohloff, 2016; Rffell et l., 2017). Potentil ntimiroil effets ginst ommon food-orne pthogens hs een shown for innmon (de Oliveir et l., 2012), lvender (Budzynsk et l., 2011), orinder nd nise (Bzrgni & Rohloff, 2016). The genus Ziziphor linopodioides hs een reported to e n effetive romti plnt with wide rnge of mediinl effets suh s ppetitive, rmintive, sedtive, stomh toni, expetornt nd ntisepti (Shhzi & Shvisi, 2016). The phytohemil studies onduted in ll prts of Z. linopodioides hrterise the presene of thymol, ɣ-terpinene, rvrol, linlool nd 1,8-ineole s ommonly predominnt EO ompounds (Behrvn et l., 2007; Aghjni et l., 2008; Shhzi & Shvisi, 2016). The genus Menth, of Lmiee fmily, omprises out speies originting in Irn, whih present numer of iologil effets. It hs severl iologil uses e.g. ntimiroil, ntioxidnt nd ntispsmodis, in good orreltion with the high ontents of phenoli ompounds (Sndsi et l., 2011). Our previous studies indited tht Z. linopodioides nd M. spit essentil oils hve strong ntiteril tivity ginst ommon food-orne pthogeni teri in food models nd in vitro ondition (Shhzi, 2015, Shhzi et l., 2016,; Shhzi & Shvisi, 2016). However, omprtive study on the ntiiofilm tivity of Z. linopodioides nd M. spit essentil oils ginst ommon food-orne pthogeni teri hs not een rried out so fr. Therefore, the im of the urrent study ws to ssess the effets of Z. linopodioides nd M. spit essentil oils ginst iofilms of six foodorne pthogens inluding L. monoytogenes, S. Typhimurium, E. oli O157:H7, B. sutilis, B. ereus nd S. ureus developed on the surfe of stinless steel oupons. 2 BJVM,, No

3 Y. Shhzi MATERIALS AND METHODS Plnt mterils The wild smples of ziziphore nd spermint were otined from nturl hitts in the Zgros region (Giln-e-Ghr, Kermnshh, western Irn) during the full flowering period (Mrh-July 2015). The plnt mterils were identified s Z. linopodioides nd M. spit y Dr. Seyed Mohmmd Msoumi, Fulty of Agriulture, Rzi University, Kermnshh, Irn. The otined plnts were shde dried t room temperture for two weeks. The dried plnts were exposed to hydrodistilltion using Clevenger-type pprtus for pproximtely 3.5 h. The isolted EOs were dried over nhydrous sodium sulfte (N 2 SO 4 ), trnsferred to seled drk vils nd stored under refrigerted ondition (4±1 ºC) until use. GC MS nlysis of Ziziphor linopodioides nd Menth spit essentil oils Gs hromtogrphy-mss spetrometry (GC-MS) nlyses of EOs were performed on Thermo Quest Finningn pprtus fitted with HP-5MS 5% phenyl methylsiloxne pillry olumn (30 m length 0.25 mm i.d. nd 0.25 µm film thikness). Helium (purity: 99.99%; flow rte 1.2 ml/min nd split rtio 1:20) ws the rrier gs. The olumn temperture ws initilly progrmmed t 50 C for 6 min nd then grdully inresed up to 265 C t 2.5 C/min. Finlly, the temperture ws inresed to 280 C t 15 C/min nd held isothermlly for 3 min. EOs nlyses were lso run on Thermo Quest Finningn oupled to mss spetrometer with the sme nlytil onditions indited ove. The MS ws run using n ionistion energy of 70 ev. The voltile hemil ompounds of the EO were identified y omprison mong their retention indies, retention indies existed in dtse, stndrd mss spetrl frgmenttion pttern (Wiley/NBS Pk v.7, 2003), nd US Ntionl Institute of Stndrds nd Tehnology. The GC pek re normlistion of the three injetions ws expressed s men perentge of the individul EO omposition. Bteril strin, ulture onditions nd inoulum preprtion Six different pthogeni teri inluding S. ureus (ATCC 6538), B. sutilis (ATCC 6633), B. ereus (ATCC 11774), L. monoytogenes (ATCC 19118), S. Typhimurium (ATCC 14028) nd E. oli O157:H7 (ATCC 10536) were purhsed from the ulture olletion of the Irnin Reserh Orgnistion for Siene nd Tehnology (IROST), Tehrn, Irn. All teril strins were ultured in Brin Hert Infusion roth (BHI; Merk, Germny) t 37 ºC for 24 h. The density of teril ultures needed for the ntiteril tests were exmined using spetrophotometer t 600 nm. The determintion of inoulum dose (7 log CFU/mL) lso ws ssessed using plte ount on BHI gr. Determintion of minimum inhiitory onentrtions (MICs) of essentil oils The minimum inhiitory onentrtions (MICs) of EOs were determined ording to the protool of Shhzi & Shvisi (2016) with minor modifitions. Different onentrtions of Z. linopodioides essentil oil (ZEO; , , , , , , nd , , µl/ml) nd M. spit essentil oil (MEO; 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, nd 0.01 µl/ml) were onstruted in BHI roth ontining 1% (v/v) dimethyl sulfoxide (DMSO). The 96-well sterile miro-dilution pltes with BJVM,, No 3

4 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. U-ottom wells were prepred y pouring 180 µl of BHI roth ontining different onentrtions of ZEO nd MEO, nd 20 µl of the investigted miroorgnisms. The lst row well ontining 180 µl BHI roth without EOs nd 20 µl of inoulum ws used s prllel positive ontrol. As negtive ontrol, the sme mount of BHI roth nd 1% (v/v) dimethyl sulfoxide (DMSO) ws lso onsidered. Contents of eh well were mixed on plte shker for 30 s nd inuted t 37 C for 24 h. The MIC ws defined s the lowest onentrtion of the ZEO or MEO t whih the miroorgnism did not demonstrte visile growth. Stinless steel oupons The flt stinless steel oupons ( m) s the experimentl surfe were prepred ording to the method previously desried y Rffell et l. (2017) with slight modifitions. Prior to use, the oupons were immersed in HCl 5N for 15 min, lened in detergent solution nd rinsed with distilled wter. The wshed stinless steel oupons were ir dried, wrpped with luminum foil nd utolved t 121 C for 15 min. Biofilm development Prepred sterile stinless steel oupons were individully immersed in 10 ml BHI roth ontining eh inoulum suspension trgeting t ell density of 7 log CFU/mL, pled in petri dishes nd inuted t room temperture (25±1 ºC) for 24 h. After inution, the stinless oupons were septilly removed with sterile foreps, wshed with sterile phosphte uffered sline (PBS; finl onentrtion of 10 mm PO 4 3, 137 mm NCl nd 2.7 mm KCl t ph 6.9) to remove untthed ells nd dried for 120 min t room temperture in io sfety inet. After drying, eh oupon ws swed with sterile polyester-tipped sws. Then, the sw ws suspended into tue ontining 1 ml uffered peptone wter nd gently vortexed for 2 min to deth teri. After gittion, ten deiml dilutions of this suspension ontining the de-tthed teri of eh treted oupon were prepred, spred on BHI gr nd inuted t 37 ºC for 24 h (Poimenidou et l., 2016; Rffell et l., 2017). Moreover, iofilms of teril pthogens developed on stinless steel surfes were nlyzed for iomss prodution using rystl violet (CV) ording to the previously method desried y Rffell et l. (2017). Briefly, eh oupon with dherent teri ws removed from petri dishes, wshed with PBS nd immersed in new dish ontining CV 0.1% (v/v) for 15 min. The oupons were wshed with PBS gin nd ir-dried. The remining CV ws dissolved in 85% ethnol for 15 min t room temperture nd, finlly, 200 μl from eh smple ws trnsferred to 96-well plte for spetrophotometry t 570 nm. All experiments were performed in triplite nd eh experiment were repeted three times. Tretments of iofilms with Ziziphor linopodioides nd Menth spit essentil oils To determine the teriidl effets of ZEO seprtely nd in omintion with MEO, the dried stinless steel oupons were dipped in 10 ml BHI roth ontining eh EO or mixture of oth t MIC onentrtion t room temperture (25 ± 1 ºC) for 1.5, 3 nd 4.5 min. At eh time point, the oupons were trnsferred into 5 ml of neutrlising solution (3% polysorte 80 v/v, 3% sponin w/v, 0.3% leithin w/v) for 2 min nd dried for 120 min t room temperture in io sfety 4 BJVM,, No

5 Y. Shhzi inet. The logrithmi redution (LR) ws estimted from the following eqution: log DP = log (N/N 0 ) = (log N) (log N 0 ); where N nd N 0 re initil dhered ells nd remining vile dhered ells following disinfetion (CFU/mL) t times t nd zero, respetively. Moreover, iomss removl tivity of the ntimiroils ginst the teril iofilms developed on stinless steel oupons ws mesured s previously desried. Sttistil nlysis All experiments were done in triplite. The nlysis ws performed using SPSS 16.0 for Windows (SPSS, Chigo, IL, USA) softwre pkge. All dt were sujeted to one-wy nlysis of vrine to determine the differenes of smples. Signifine level ws onsidered P<0.05 for ll experimentl dt. RESULTS GC MS nlysis of Ziziphor linopodioides nd Menth spit essentil oils The hemil ompositions of ZEO nd MEO re presented in Tles 1 nd 2, respetively. Twenty-four ompounds in the ZEO were identified, representing 99.65% of the totl oil. The mjor ompounds de- Tle 1. Composition of essentil oil of Ziziphor linopodioides identified y GC-MS No. Compound nme Composition % Retention time (min.) 1 α-thujene α-pinene Cmphene β-pinene Oten-3-ol Myrene α-phellndrene α-terpinene p-cymene Limonene β-phellndrene γ-terpinene is-sinene hydrte Terpinolene Linlool Borneol Terpinene-4-ol α-terpineol Crvrol, methyl ether Thymol Crvrol E-Cryophyllene Spthulenol Cryophyllene oxide Other 0.08 Totl BJVM,, No 5

6 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. Tle 2. Composition of essentil oil of Menth spit identified y GC-MS No. Compound nme Composition % Retention time (min.) 1 β-myrene Limonene γ-terpinene Menthone Menthol Terpinen-4-ol α-terpinol Dihydrorveol is-dihydrorveol Dihydrorvone trns-crveol Crvone Dihydrorvyl ette L-rveol β-bouronene trns-cryophyllene γ -Amorphene α-amorphene Others 0.11 Totl 100 teted in the ZEO were rvrol (65.22%), thymol (19.51%), p-ymene (4.86%) nd ɣ-terpinene (4.63%). As it n e seen in Tle 2, the GC- MS nlysis of MEO indited the presene of eighteen ompounds representing 99.89% of the totl oil. The MEO ws minly omposed from rvone (78.76%), limonene (11.50%) nd β-ouronene (11.23%). Antiteril tivity of Ziziphor linopodioides nd Menth spit essentil oils The first prt of the urrent study ws onduted to investigte the in vitro ntiteril tivity of ZEO nd MEO ginst plnktoni ells of ommon foodorne pthogeni teri inluding S. ureus, B. sutilis, B. ereus, L. monoytogenes, S. Typhimurium nd E. oli O157:H7. Bsed on the results of the present study (Tle 3), the ntiteril tivity differed signifintly etween the two EOs (P<0.05). As it n e seen, the MIC vlues of ZEO nd MEO ginst investigted miroorgnisms were nd µl/ml, respetively. Biofilm formtion The ility of S. Typhimurium, E. oli O157:H7, L. monoytogenes, S. ureus, B. sutilis nd B. ereus to dhere nd form iofilms on stinless steel surfes ws determined y CFU/m 2 enumertion nd iomss nlysis (Tle 4). The rnk order of iofilm prodution were s follow: S. Typhimurium > E. oli O157:H7 > S. ureus > L. monoytogenes > B. sutilis > B. ereus. 6 BJVM,, No

7 Y. Shhzi Tle 3. Minimum inhiitory onentrtions (MICs) of Ziziphor linopodioides nd Menth spit essentil oils ginst seleted teril pthogens Bteri MIC (µl/ml) Z. linopodioides M. spit Z. linopodioides + M. spit Stphyloous ureus Billus sutilis Billus ereus Listeri monoytogenes Slmonell Typhimurium Esherihi oli O157:H Tle 4. Colony forming unit ounts nd iomss of teril iofilms formed on stinless steel oupons Bteri Plte ount gr (CFU/m 2 ) Biomss nlysis (OD) Stphyloous ureus 5.21± ±0.01 Billus sutilis 4.23±0.02 e 1.38±0.01 e Billus ereus 4.17±0.01 f 1.21±0.02 f Listeri monoytogenes 4.62±0.05 d 1.41±0.03 d Slmonell Typhimurium 5.75± ±0.01 Esherihi oli O157:H7 5.31± ±0.02 Dt represent the men vlues otined in three independent experiments performed in triplite; - Different letters indite signifint differenes mong the different teril pthogens (P<0.05). Effetiveness of Ziziphor linopodioides nd Menth spit essentil oils on teril pthogen iofilms The nti-iofilm effets of disinfetnt solutions sed on ZEO seprtely nd in omintion with MEO ginst S. ureus, B. sutilis, B. ereus, L. monoytogenes, S. Typhimurium nd E. oli O157:H7 on the stinless steel oupons, with ontt times of 1.5, 3 nd 4.5 min, re presented in Fig. 1A-1F. Bsed on our findings (Fig. 1A-1F), there ws signifint differene etween the popultion of ll investigted pthogens on the stinless steel oupons treted with ZEO nd MEO (P<0.05). The nti-iofilm tivity of the disinfetnt solutions sed on ZEO nd MEO n e expressed s logrithmi redution (LR) etween initil dhered ells nd remining vile dhered ells following disinfetion. Therefore, in ll teril iofilms treted with the omintion of ZEO nd MEO LRs higher thn 2 log CFU/m 2 were oserved. The perentges of iofilm removl tivities of ZEO nd MEO on the stinless steel oupons with ontt times of 1.5, 3 nd 4.5 min re presented in Tle 5. Bsed on our findings, ZEO showed signifintly higher iofilm removl tivity ompred to MEO (P<0.05). Indeed, regrding the disinfetnt solution sed on ZEO, the highest iofilm removl tivity ws found for B. ereus, followed y B. sutilis, L. monoytogenes, S. ureus, E. oli O157:H7 nd S. Typhimurium, respetively. The order of io- BJVM,, No 7

8 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. Stphyloous ureus (log CFU/m2) 6 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 0 min 1.5 min 3 min 4.5 min Control MEO ZEO ZEO+MEO Billus sutilis (log CFU/m2) 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 0 min 1.5 min 3 min 4.5 min Control MEO ZEO ZEO+MEO A B Billus ereus (log CFU/m2) 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 min 1.5 min 3 min 4.5 min Listeri monoytogenes (log CFU/m2) 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 d d 0 min 1.5 min 3 min 4.5 min 0 Control MEO ZEO ZEO+MEO 0 Control MEO ZEO ZEO+MEO Slmonell Typhimurium (log CFU/m2 6,5 6 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 d C d Control MEO ZEO ZEO+MEO 0 min 1.5 min 3 min 4.5 min Esherihi oli O157:H7 (log CFU/m2) 6 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 d D Control MEO ZEO ZEO+MEO E F Fig. 1. Antiteril tivity of Ziziphor linopodioides essentil oil (ZEO) seprtely nd in omintion with Menth spit essentil oil (MEO) ginst iofilms of Stphyloous ureus (A), Billus sutilis (B), Billus ereus (C), Listeri monoytogenes (D), Slmonell Typhimurium (E) nd Esherihi oli O157:H7 (F) developed on stinless steel oupons. Dt represent the men vlues otined in three independent experiments performed in triplite; -d for eh tretment, different letters indite signifint differenes in the perentges of iofilm removl tivities on the stinless steel oupons mong ontt times of 1.5, 3 nd 4.5 min (P<0.05). d 0 min 1.5 min 3 min 4.5 min film removl tivity of disinfetnt solution sed on MEO ws: B. ereus > B. sutilis > L. monoytogenes > S. ureus > E. oli O157:H7 > S. Typhimurium. Dt nlysis reltive to the iofilm removl tivity of the disinfetnt solu- 8 BJVM,, No

9 Y. Shhzi Tle 5. Perentge of teril iofilm removl y Ziziphor linopodioides (ZEO) nd Menth spit (MEO) essentil oils lone nd in omintions Bteri Stphyloous ureus Billus sutilis Billus ereus Listeri monoytogenes Slmonell Typhimurium Esherihi oli O157:H7 Biofilm removl (%) of the essentil oils (men ± sd) Contt time (min) Z. linopodioides M. spit Z. linopodioides + M. spit (±0.3) B (±0.2) C (±0.1) A (±0.3) B (±0.3) C 100 (±0.0) A (±0.1) B (±0.6) C 100 (±0.0) A (±0.2) B (±0.1) C (±0.1) A (±0.1) B (±0.8) C 100 (±0.0) A (±0.4) B (±0.2) C 100 (±0.0) A (±0.3) B (±0.3) C (±0.2) A (±0.4) B (±0.5) C 100 (±0.0) A (±0.1) B (±0.1) C 100 (±0.0) A (±0.5) B (±0.2) C (±0.7) A (±0.1) B (±0.5) C 100 (±0.0) A (±0.2) B (±0.6) C 100 (±0.0) A (±0.2) B 5.73 (±0.2) C (±0.4) A (±0.1) B (±0.1) C 100 (±0.0) A (±0.1) B (±0.2) C 100 (±0.0) A (±0.1) B 9.03 (±0.2) C (±0.1) A (±0.1) B (±0.5) C 100 (±0.0) A (±0.4) B (±0.4) C 100 (±0.0) A Dt represent the men vlues otined in three independent experiments performed in triplite. A-C Mens with different pitl letters in the sme row re signifintly different (P < 0.05). - For eh terium, mens with different lowerse letters in the sme olumn re signifintly different (P < 0.05). BJVM,, No 9

10 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. tion sed on the omintion of ZEO nd MEO showed signifint iofilm removl ility of the disinfetnt solution ginst ll teri, with perentges higher thn those of otined with eh single EOs (P<0.05). With respet to the ontt times utilized, the numer of surfe-dhered ells ws signifintly deresed fter 1.5, 3 nd 4.5 min of exposure to the disinfetnt solutions sed on ZEO seprtely nd in omintion with MEO (P<0.05). The remined teril popultion fter 3 nd 4.5 min of ontt were signifintly lower thn those tht remined fter 1.5 min of tretment (P 0.05). Moreover, the most drsti redution of teril popultion ws oserved fter 3 nd 4.5 min tretment with the disinfetnt solutions sed on EOs (P 0.05). DISCUSSION The high perentges of rvrol nd thymol identified Z. linopodioides s good soure of these vlule ompounds. These two ompounds together onstitute 84.73% of the totl oil omposition of Z. linopodioides. Our results greed with nother study whih reported rvrol nd thymol s the mjor ompounds (72.5%) of ZEO (Aghjni et l., 2008). However, ZEO from different ultivrs nd lotions were found to ontin pulegone, terpineol, methyl ette, isoneomenthol nd 1, 8-ineole (Mortez- Semnni et l., 2005; Behrvn et l., 2007; Sonoli et l., 2010; M et l., 2016). The omposition ontents of MEO in the urrent study is in good greement with previous reports (Mhoui & Hghi, 2008; Govindrjn et l., 2012). Generlly, the hemil ompositions differenes of EOs my e explined with vriility of plnt speies, geogrphil, limte nd sesonl onditions (Shhzi & Shvisi, 2016). Bsed on the results of the present study (Tle 3), the ntiteril tivity differed signifintly etween the two EOs (P<0.05). The signifint differene in the ntimiroil tion of EOs is relted to the vriility in the nture nd onentrtion of min groups of hemil ompositions (Kkei & Shhzi, 2016). The ntiteril mehnism of ZEO ws suggested to e due to the -OH groups loted t the met nd ortho positions in thymol nd rvrol, respetively, whih re le to intert with ytoplsmi memrne of teril ells. It is elieved tht this phenomenon n disrupt the teril phospholipid memrne resulting in inhiition of eletron trnsport, protein trnslotion, phosphoryltion nd other enzymti tivity nd thus leding to ell deth (Gywli & Irhim, 2014). Mhoui & Hghi (2008) hve indited tht rvone s the mjor ompounds of MEO used extensive dmge of phospholipid ilyer struture, redued the ph grdient ross the teril memrne nd susequently inhiited the teril rrier funtion. However, it hs een reported tht numer of ompounds in reltively low onentrtions suh s p- ymene, ɣ-terpinene, α-pinene, β-ouronene, α-terpinol nd α-terpinene ould lso e expeted to enhne the ntimiroil tivity of the EOs (Lv et l., 2011). As shown in Tle 3, it is ler tht Grm-positive teri (S. ureus, B. sutilis, B. ereus nd L. monoytogenes) were more sensitive thn Grm-negtive teri (S. Typhimurium nd E. oli O157:H7). The most prole reson for the differenes in miroorgnism s suseptiility to EOs might e ttriuted to the outer ytoplsmi memrne surrounding the thin peptidoglyn struture 10 BJVM,, No

11 Y. Shhzi of Grm-negtive teri, whih restrits the diffusion of hydrophoi ompounds through its lipopolyshride overing (Bzrgni & Rohloff, 2016). In this study, the rnk order of iofilm prodution ws s followed: S. Typhimurium > E. oli O157:H7 > S. ureus > L. monoytogenes > B. sutilis > B. ereus. The prole reson ehind onsiderle differenes in iofilm formtion y investigted teri n e ttriuted to differenes in moility, inherent ell surfe hrteristis nd prodution of psulr polyshride/dhesion mong the miroil speies (Rivs et l., 2010). Aording to the literture review performed y Kusumningrum et l. (2003), the presene of extrellulr ppendges suh s flgell nd pili, intertions involved in ell ell ommunition nd prodution of extrellulr polymeri sustnes re the most importnt ftors whih ffet the ehviour of teril ells on stinless steel surfes. Most of findings reported from other studies re not omprle sine different methodology nd environmentl onditions suh s room temperture nd type of teril strins n remrkly influene the reovery rtios of miroorgnisms on stinless steel surfes (Perez-Rodriguez et l., 2013). However, Ryu et l. (2004) reported tht pproximtely 5.30 log CFU/m 2 of E. oli O157:H7 were found dherent on stinless steel fter 24 h inution t room temperture with 7 log CFU/mL teril suspension, whih is in good greement with our findings. In nother study, Be et l. (2012) demonstrted tht S. Typhimurium ould dhere to stinless steel surfe t rte of 3 log CFU/m 2, onsidering n initil ontmintion of 5 log CFU/mL. de Oliveir et l. (2010) inoulted 8 log CFU/mL of E. oli nd L. monoytogenes onto stinless steel surfes nd formed iofilms, followed y inution t room temperture for 24 h. Aording to their results, 4.86 nd 5.64 log CFU/m 2 of forementioned teri in iofilms were reovered. Bsed on our findings (Fig. 1A-1F), there ws signifint differene etween the popultion of ll investigted pthogens on the stinless steel oupons treted with ZEO nd MEO (P<0.05). Differenes in the ntiteril effets etween ZEO nd MEO n e ttriuted to the nture nd onentrtion of their tive hemil ompositions (Vázquez-Sánhez et l., 2015). Despite the lk of ny onlusive dt out the nti-iofilm tivity of ZEO, some studies hve showed its powerful ntiteril tivity ginst severl importnt food-orne pthogens inluding B. sutilis, B. ereus, S. ureus, L. monoytogenes, S. Typhimurium nd E. oli O157:H7 (Aghjni et l., 2008; Kkei & Shhzi, 2016; Shhzi & Shvisi, 2016; Mohei & Shhzi, 2017). Moreover, the disinfetnt solution sed on MEO ould modertely inhiit the growth of sessile ells of ll tested strins (Fig. 1A-1F). Vlerino et l. (2012) exmined the nti-iofilm tivity of MEO ginst S. enteri serotype Enteritidis on stinless steel surfe fter 10, 20 nd 40 min. They reported tht ten minutes of MEO disinfetnt solution ontt signifintly redued dhered teril popultion (P<0.05). The sme results were lso reported in the study of Sndsi et l. (2011) who investigted the effet of MEO ginst iofilm formtion y Pseudomons eruginos nd Cndid lins. As presented in our findings, the most drsti redution of teril popultion ws oserved fter 3 nd 4.5 min tret- BJVM,, No 11

12 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. ment with the disinfetnt solutions sed on EOs (P 0.05). Sine tive groups of EOs suh s -OH n intert with the teril ell memrne nd susequently use the lekge of ellulr omponents, it n e onluded tht the long ontt time etween the miroorgnism nd the disinfetnt solutions sed on EOs led to extensive loss of ell ontents or ritil moleules nd ions (Vlerino et l., 2012; Gywli & Irhim, 2014). Despite the ft tht some loss in the mount of ell ontent is tolerted y the teri without loss of their viility, extensive loss of ell ontent or essentil moleules nd ions will led to teril ell deth (de Oliveir et l., 2010). The results of the present study demonstrted tht tretments of stinless steel surfes for 3 nd 4.5 min with the disinfetnt solution sed on the omintion of ZEO nd MEO ws suffiient for totl removl iofilm formed y ll tested pthogens (Tle 5). Our in vitro previous study hs proved tht the omintion of ZEO nd MEO displyed synergisti tivity ginst B. sutilis, B. ereus, S. ureus, L. monoytogenes, S. Typhimurium nd E. oli O157:H7 (Shhzi & Shvisi, 2016). It hs een shown tht synergisti effets of EOs ffet miroil ells y vrious ntimiroil mehnisms, inluding sequentil inhiition of ommon iohemil pthwy, disrupting of protetive enzymes nd inresing the numer nd size of pores reted in phospholipid ilyer of the ell memrne (Mohei & Shhzi, 2017). In ordne with our findings, de Oliveir et l. (2010) reported tht the disinfetnt solutions sed on omintion of Cymopogon itrtus nd Cymopogon nrdus EOs ould ompletely remove iofilm of L. monoytogenes on the stinless steel surfe. The Amerin Puli Helth Assoition (APHA) hs indited tht stndrd physil or hemil disinfetnt must e ple of erditing pthogeni teri nd deresing the numer of deteriorting mesophili eroi miroorgnisms to eptle levels of 0.3 log CFU/m 2 or 2 log CFU/utensil t the end of the disinfetion proess (Anonymous, 1992). Therefore, the disinfetnt solution sed on the omintion of ZEO nd MEO, tht fulfilled the proposed reommendtion, n e used s n pproprite nturl disinfetnt to ontrol iofilm formtion in food proessing filities nd utensils. CONCLUSION The results of the present study indited tht ZEO nd MEO presented good soure of iotive ompounds suh s rvrol, thymol, rvone, limonene nd β-ouronene. These sustnes were mostly found in the plnt speies, ut further reserh is required to identify the phytohemil ompounds of the MEO nd ZEO responsile for their ntiiofilm effets. To our knowledge, this is the first time the ntiiofilm tivity of ZEO seprtely nd in omintion with MEO hs een reported ginst iofilms formed y forementioned pthogens. Bsed on our findings, ZEO nd MEO re new lterntive ompounds to snitise industril stinless steel surfes ontminted with S. ureus, B. sutilis, B. ereus, L. monoytogenes, S. Typhimurium nd E. oli O157:H7. ACKNOWLEDGEMENTS The uthor knowledges Rzi University for the use of their filities nd instrumenttions. 12 BJVM,, No

13 Y. Shhzi REFERENCES Aghjni, Z., F. Assdin, S. Msoudi, F. Chlin, A. Esmeili, M. Ttei- Anrki & A. Rustiyn, Chemil omposition nd in vitro ntiteril tivities of the oil of Ziziphor linopodioides nd Z. pitt susp. pitt from Irn. Chemistry of Nturl Compounds, 44, Anonymous, Compendium of methods for the miroiologil exmintion of foods. Hnover: EPS Group. Be, Y. M., S. Y. Bek & S. Y. Lee, Resistne of pthogeni teri on the surfe of stinless steel depending on tthment form nd effiy of hemil snitizers. Interntionl Journl of Food Miroiology, 153, Bzrgni, M. M. & J. Rohloff, Antiiofilm tivity of essentil oils nd plnt extrts ginst Stphyloous ureus nd Esherihi oli iofilms. Food Control, 61, Behrvn, J., M. Rmezni, M. Hssnzdeh, M. Eskndri, J. Ksin & Z. Seti, Composition, ntimyoti nd ntiteril tivity of Ziziphor linopodioides Lm. essentil oil from Irn. Journl of Essentil Oil Bering Plnts, 10, Budzynsk, A., M. Wiekowsk-Szkiel, B. Sdowsk, D. Klem & B. Rózlsk, Antiiofilm tivity of seleted plnt essentil oils nd their mjor omponents. Polish Journl of Miroiology, 60, Csrin, L. S., A. Brndelli, F. de Oliveir Csrin, P. A. Sove, C. H. Wnke & E. C. Tondo, Adhesion of Slmonell Enteritidis nd Listeri monoytogenes on stinless steel welds. Interntionl Journl of Food Miroiology, 191, Chen, D., T. Zho & M. P. Doyle, Control of pthogens in iofilms on the surfe of stinless steel y levulini id plus sodium dodeyl sulfte. Interntionl Journl of Food Miroiology, 207, 1 7. de Oliveir, M. M. M., D. F. Brugner, M. ds Grçs Crdoso, E. Alves & R. H. Pioli, Disinfetnt tion of Cymopogon sp. essentil oils in different phses of iofilm formtion y Listeri monoytogenes on stinless steel surfe. Food Control, 21, de Oliveir, M. M. M., D. F. Brugner, J. A. do Nsimento, N. N. Btist & R. H. Pioli, Cinnmon essentil oil nd innmldehyde in the ontrol of teril iofilms formed on stinless steel surfes. Europen Food Reserh nd Tehnology, 234, Govindrjn, M., R. Sivkumr, M. Rjeswri & K. Yoglkshmi, Chemil omposition nd lrviidl tivity of essentil oil from Menth spit (Linn.) ginst three mosquito speies. Prsitology Reserh, 110, Gywli, R. & S. A. Irhim, Nturl produts s ntimiroil gents. Food Control, 46, Kkei, S. & Y. Shhzi, Effet of hitosn-geltin film inorported with ethnoli red grpe seed extrt nd Ziziphor linopodioides essentil oil on survivl of Listeri monoytogenes nd hemil, miroil nd sensory properties of mined trout fillet. LWT-Food Siene nd Tehnology, 72, Kim, M., S. Y. Prk & S. D. H, Synergisti effet of omintion of ultrviolet C irrdition nd sodium hypohlorite to redue Listeri monoytogenes iofilms on stinless steel nd eggshell surfes. Food Control, 70, Kud, T., T. Koyngi, G. Shit, H. Tkhshi & B. Kimur, Effet of rrot residue on the desition nd disinfetnt resistnes of food relted pthogens dhered to stinless steel surfes. LWT-Food Siene nd Tehnology, 74, Kusumningrum, H., G. Rioldi, W. Hzeleger & R. Beumer, Survivl of foodorne pthogens on stinless steel surfes nd BJVM,, No 13

14 Antiteril effets of Ziziphor linopodioides nd Menth spit essentil oils ginst ommon. ross-ontmintion to foods. Interntionl Journl of Food Miroiology, 85, Lv, F., H. Ling, Q. Yun & C. Li, In vitro ntimiroil effets nd mehnism of tion of seleted plnt essentil oil omintions ginst four food-relted miroorgnisms. Food Reserh Interntionl, 44, M, B.X., X. Q. Bn, J. S. He, B. Hung, H. Zeng, J. Tin, Y. X. Chen & Y. W. Wng, Antifungl tivity of Ziziphor linopodioides Lm. essentil oil ginst Slerotini slerotiorum on rpeseed plnts (Brssi mpestris L.). Crop Protetion, 89, Mhoui, M. & G. Hghi, Antimiroil tivity nd hemil omposition of Menth pulegium L. essentil oil. Journl of Ethnophrmology, 119, Mohei, E. & Y. Shhzi, Applition of hitosn nd geltin sed tive pkging films for peeled shrimp preservtion: A novel funtionl wrpping design. LWT-Food Siene nd Tehnology, 76, Prt A, Mortez-Semnni, K., M. Seedi & G. Eslmi, Essentil oil omposition of Ziziphor linopodioides Lm. from Irn. Journl of Essentil Oil Bering Plnts, 8, Nm, H., H. S. Seo, J. Bng, H. Kim, L. R. Beuht & J. H. Ryu, Effiy of gseous hlorine dioxide in intivting Billus ereus spores tthed to nd in iofilm on stinless steel. Interntionl Journl of Food Miroiology, 188, Ouli, F. A., I. Al Kss, B. Cudenne, M. Adllh, F. Bendli, D. Sdoun, N. E. Chihi & D. Drider, Identifition of ltoilli with inhiitory effet on iofilm formtion y pthogeni teri on stinless steel surfes. Interntionl Journl of Food Miroiology, 191, Perez-Rodriguez, F., G. Posd-Izquierdo, A. Vlero, R. Grí-Gimeno & G. Zurer, Modelling survivl kinetis of Stphyloous ureus nd Esherihi oli O157:H7 on stinless steel surfes soiled with different sustrtes under stti onditions of temperture nd reltive humidity. Food Miroiology, 33, Poimenidou, S. V., M. Chrysdkou, A. Tzkoniti, V. C. Bikouli, G. J. Nyhs & P. Skndmis, N Vriility of Listeri monoytogenes strins in iofilm formtion on stinless steel nd polystyrene mterils nd resistne to pereti id nd quternry mmonium ompounds. Interntionl Journl of Food Miroiology, 237, Rffell, C., L. Csettri, L. Fgioli, M. Cespi, G. Bonuin & W. Bffone, Ativity of essentil oil-sed miroemulsions ginst Stphyloous ureus iofilms developed on stinless steel surfe in different ulture medi nd growth onditions. Interntionl Journl of Food Miroiology, 241, Rivs, L., M. J. MDonnell, C. M. Burgess, M. O'Brien, A. Nvrro-Vill, S. Fnning & G. Duffy, Inhiition of veroytotoxigeni Esherihi oli in model roth nd rumen systems y rvrol nd thymol. Interntionl Journl of Food Miroiology, 139, Ryu, J. H., H. Kim, J. Frnk & L. Beuht, Atthment nd iofilm formtion on stinless steel y Esherihi oli O157:H7 s ffeted y urli prodution. Letters in Applied Miroiology, 39, Sndsi, M., C. Leonrd, S. Vn Vuuren & A. Viljoen, Peppermint (Menth piperit) inhiits miroil iofilms in vitro. South Afrin Journl of Botny, 77, Shlisselerg, D. B. & S. Yron, The effets of stinless steel finish on Slmonell Typhimurium tthment, iofilm formtion nd sensitivity to hlorine. Food Miroiology, 35, BJVM,, No

15 Y. Shhzi Shhzi, Y., Chemil omposition nd in vitro ntiteril tivity of Menth spit essentil oil ginst ommon food-orne pthogeni teri. Journl of Pthogens, 2015, , Shhzi, Y. & N. Shvisi, Intertions of Ziziphor linopodioides nd Menth spit essentil oils with hitosn nd iprofloxin ginst ommon food-relted pthogens. LWT-Food Siene nd Tehnology, 71, Shhzi, Y., N. Shvisi & E. Mohei, Effets of Ziziphor linopodioides essentil oil nd nisin, oth seprtely nd in omintion, to extend shelf life nd ontrol Esherihi oli O157:H7 nd Stphyloous ureus in rw eef ptty during refrigerted storge. Journl of Food Sfety, 36, Shhzi, Y., N. Shvisi & E. Mohei, Potentil pplition of Ziziphor linopodioides essentil oil nd nisin s nturl preservtives ginst Billus ereus nd Esherihi oli O157:H7 in ommeril rley soup. Journl of Food Sfety, 36, Sonoli, A., M. Atri, & S. Shfiei, Intrspeifi vriility of the essentil oil of Ziziphor linopodioides ssp. rigid from Irn. Chemistry & Biodiversity, 7, Vlerino, C., T. L. C. De Oliveir, S. M. De Crvlho, M. ds Grçs Crdoso, E. Alves & R. H. Pioli, The snitizing tion of essentil oil-sed solutions ginst Slmonell enteri serotype Enteritidis S64 iofilm formtion on AISI 304 stinless steel. Food Control, 25, Vázquez-Sánhez, D., M. L. Co & J. J. Rodríguez-Herrer, Antimiroil tivity of essentil oils ginst Stphyloous ureus iofilms. Food Siene nd Tehnology Interntionl, 21, Pper reeived ; epted for pulition Correspondene: Dr. Ysser Shhzi Deprtment of Food Hygiene nd Qulity Control, Fulty of Veterinry Mediine, Rzi University, Kermnshh, Irn emil: ysser.shhzi@yhoo.om, y.shhzi@rzi..ir BJVM,, No 15