Influence of Ethylenediaminetetraacetic Acid (EDTA) on the Ability of Fatty Acids to Inhibit the Growth of Bacteria Associated with Poultry Processing

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1 Interntionl Journl of Poultry Siene (7): -4, ISSN Asin Network for Sientifi Informtion, Influene of Ethylenediminetetreti Aid (EDTA) on the Aility of Ftty Aids to Inhiit the Growth of Bteri Assoited with Poultry Proessing A. Hinton Jr. nd K.D. Ingrm Poultry Proessing Unit nd Swine Physiology Reserh Unit, Agriulturl Reserh Servie, United Sttes Deprtment of Agriulture, 9 College Sttion Rod, Russell Reserh Center, Athens, GA, USA 36 Astrt: The effet of Ethylenediminetetreti Aid (EDTA) on the teriidl tivity of Ftty Aids (FA) ws exmined. A. M onentrtion of proi, pryli, pri, or luri id in. M Potssium Hydroxide (KOH) ws supplemented with,, or mm of EDTA nd djusted to ph. with itri id. FA-KOH-EDTA 6 ws dded to wells in gr seeded with fu/ml of Ainetoter loetius, Aeromons hydrophil, Cmpyloter jejuni, Enteroous felis, Esherihi oli, Listeri monoytogenes, Pseudomons fluoresens, Slmonell typhimurium, or Stphyloous simulns. Agr pltes were inuted eroilly t 3 C for 4 h, exept for C. jejuni pltes whih were inuted miroerophililly t 4 C for 48 h nd zones of inhiition round the wells were mesured. Results indited tht lthough proi id-koh inhiited growth of C. jejuni only, proi id-koh supplemented with EDTA produed signifintly (p<.) lrger zones of C. jejuni s well s zones of inhiition of A. loetius, E. felis nd P. fluoresens. Cpryli id-koh produed zones of inhiition of ll isoltes exept A. loetius, L. monoytogenes nd S. simulns, ut supplementtion with EDTA produed zones of inhiition of the 3 isoltes in ddition to inreses in the size of zones of inhiition of E. felis nd Slmonell Typhimurium. Cpri id-koh nd luri id-koh inhiited ll isoltes nd supplementing oth mixtures with EDTA generlly produed lrger zones of inhiition. Findings indite tht the ddition of EDTA to formultions of FA-KOH my inrese the ility of these snitizers to redue ontmintion of poultry proessing opertions. Key words: EDTA, ftty ids, teriidl, gr diffusion ssy, poultry proessing INTRODUCTION Some ftty ids found in niml nd vegetle oils possess ntimiroil tivity whih n e used to inhiit the growth of miroorgnisms ssoited with food produts (Kr et l., 977). The gr diffusion ssy hs previously een used to demonstrte the ntiteril tivity of lkline slts of proi, pryli, pri, luri nd myristi ids towrds severl teri isolted from proessed poultry (Hinton nd Ingrm, ). Beuse of their sop-like qulities, lkline slts of ftty ids lso t s surftnts tht my e used to wsh wy miroorgnisms from ontminted surfes (Hinton et l., 9). Effetive formultions of teriidl surftnts my serve s sis for the development of lterntives to hemil snitizers (Oyrzl, ) urrently used in poultry proessing. Metl heltors re often dded to formultions of sops nd detergents to inrese the ility of these lensers to remove dirt nd deris from surfes (Ngrjn nd Pine, 984). When dded to surftnt formultions, hrdness therey deresing lensing tivity of wter (Kovh, 7). Ethylenediminetetreti Aid (EDTA) is helting gent often used s detergent uilder in surftnt formultions. EDTA lso possesses teriidl tivity (Chew et l., 98), whih n lso inhiit the growth of severl teri found in poultry proessing opertions) (Hinton nd Ingrm, ). Furthermore, EDTA n inrese the sensitivity of some teri to other teriides (Phillips nd Duggn, ; Russell nd Furr, 977) y inresing the permeility of teril ellulr memrnes (Gry nd Wilkinson, 96; Lieve, 968; Wilkinson, 967). The purpose of the urrent study ws to exmine the ility of EDTA to inrese the ntiteril tivity of lkline slts of ftty ids towrds teri ssoited with poultry proessing. MATERIALS AND METHODS Growth of teril ultures nd inoultion of gr medi: Proedures used for the growth nd preprtion metl heltors t s detergent uilders tht sequester of suspensions of Ainetoter loetius, or preipitte minerls, suh s lium nd Aeromons hydrophil, Cmpyloter jejuni 48, mgnesium, whih n e dissolved in wter. High Enteroous felis, Esherihi oli, Listeri onentrtions of these minerls n inrese wter monoytogenes, Pseudomons fluoresens, Slmonell Corresponding Author: A. Hinton Jr., Poultry Proessing Unit nd Swine Physiology Reserh Unit, Agriulturl Reserh Servie, United Sttes Deprtment of Agriulture, 9 College Sttion Rod, Russell Reserh Center, Athens, GA, USA 36

2 Int. J. Poult. Si., (7): -4, typhimurium nd Stphyloous simulns ultures inhiition of C. jejuni thn proi id-koh (Tle ). were previously desried (Hinton nd Ingrm, ). Furthermore, proi id-koh supplemented with Fresh ultures of the teril isoltes were used to mm EDTA produed signifintly lrger zones of prepre inoulted gr medi y dding suspensions inhiition of C. jejuni nd P. fluoresens thn mixtures of eh ulture, exept for C. jejuni, to seprte liquots supplemented with only mm of EDTA, s well s of sterile Difo Trypti Soy Agr (Beton Dikinson nd signifint zones of inhiition of A. loetius nd E. Co., Sprks, MD) whih hd een tempered to C. felis. The extensively studied sensitivity of some Suspensions of C. jejuni ultures were dded to Pseudomons speies to the ntiteril tivity of tempered Blood Agr (Beton Dikinson nd Co.) EDTA (Wilkinson, 967) hs een linked to the high supplemented with 7% lysed horse lood (Lmpire onentrtion of phosphorous (Gry nd Wilkinson, Biologil Lortories, In., Pipersville, PA). Eh 96) nd multivlent tions (I. e. mgnesium, lium 6 inoulted gr medi ontined /olony-forming- nd zin) (Asell nd Egon, 966) found in the ellulr units (fu) of one of the isoltes/ml. memrne of pseudomonds. All isoltes inhiited y proi id-koh-edta were Grm negtive rods, Preprtion of ftty id-edta mixtures: Alkline slts exept for the Grm positive ous, E. felis. EDTA of proi (hexnoi), pryli (otnoi), pri hs een shown to e n effetive disinfetnt tht (denoi) nd luri (dodenoi) ids (ll otined prevents infetions y E. felis when used s dentl from Sigm Chemil Co; St. Louis, MO) were prepred irrignt during root nl surgery (Bulio et l., 6). y dissolving. M of eh ftty id (FA) in seprte Previous reserh hs illustrted tht C. jejuni is the volumes of. M potssium hydroxide (KOH). The FA- only isolte used in this study tht is suseptile to the KOH mixtures were then supplemented with,, or teriidl tivity of proi id (Hinton nd Ingrm, mm of ethylenediminetetreti id, dipotssium slt ) while nother study indited tht exposure to dihydrte (EDTA) (Sigm Chemil Co.) nd djusted to mm EDTA produed signifint zones of inhiition of ph. with itri id (Sigm Chemil Co.). Prepred most of the teri used in the present study (Hinton solutions were filter sterilized using. µm filters (Nlge nd Ingrm, ). No zones of inhiition of E. oli, L. Nun Interntionl, Rohester, NY) nd. ml of the monoytogenes, Slmonell typhimurium nd S. solutions were dded to 8 mm wells tht hd een simulns were produed y mixtures of proi idpunhed in the gr. All pltes were inuted KOH supplemented with mm EDTA, however. eroilly t 3 C for 8-4 h, exept for C. jejuni pltes Cpryli id-koh lone inhiited growth of A. whih were inuted miroerophililly t 4 C for 48 hydrophil, C. jejuni, E. felis, E. oli, P. fluoresens h. Zones of inhiition round the wells were then nd Slmonell typhimurium growth, while pryli idmesured long single plne with Trele Cron KOH supplemented with mm EDTA produed zones of Fier Digitl Clipers (Fisher Sientifi, In., Pittsurg, inhiition of A. loetius nd E. felis tht were PA, USA) (Hinton nd Ingrm, ). Experiment ws signifintly lrger thn zones produed y pryli idreplited times. KOH (Tle ). Furthermore, the size of the zones of inhiition of L. monoytogenes formed y pryli idmixture Sttistis: Sttistil nlyses of differenes in the size KOH with mm EDTA were signifintly lrger thn of zones of inhiition of eh teril isolte produed zones of inhiition produed y pryli id-koh. y FA-KOH-EDTA were performed using the GrphPd Additionlly, the zones of inhiition of A. loetius, E. InStt version 3. for Windows 9 (GrphPd felis, Slmonell typhimurium nd S. simulns Softwre, Sn Diego, CA). One-wy Anlysis of Vrine formed y pryli id-koh with mm EDTA were (ANOVA) with Tukey-Krmer Multiple Comprison tests signifintly lrger thn zones of inhiition produed y ws performed to determine signifint differenes in pryli id-koh with mm EDTA. Although pryligroup mens. The P vlue for ll ANOVA tests ws < KOH without EDTA inhiited the growth of A. hydrophil,.. C. jejuni, E. oli nd P. fluoresens, supplementing this with EDTA did not result in lrger zones of RESULTS AND DISCUSSION Previous studies hve demonstrted tht solutions of inhiition of these isoltes. A previous study indited tht the Grm negtive isoltes, C. jejuni, E. oli nd P. EDTA (Hinton nd Ingrm, ) nd solutions of fluoresens exhiited reltively high degree of vrious FA-KOH (Hinton nd Ingrm, ) n inhiit sensitivity towrds the ntiteril tivity of pryliteri growth; however, results of the present study KOH (Hinton nd Ingrm, ); therefore, it is possile indite tht FA-KOH-EDTA solutions my possess tht sine pryli-koh nd EDTA use lysis of greter ntiteril thn FA-KOH mixtures. Cproi teril ellulr memrnes ntiteril synergisms id-koh exhiited teriidl tivity towrds C. jejuni etween these two gents might not e evident in highly only; however, proi id-koh mixtures supplemented suseptile isoltes. with mm EDTA lso produed zones of inhiition of P. Cpri-KOH (Tle 3) nd luri-koh (Tle 4) fluoresens in ddition to signifintly lrger zones of produed zones of inhiition of ll teril isoltes.

3 Int. J. Poult. Si., (x): xx-xx, Tle : Size (mm) of zones of inhiition of teril isoltes y proi id-koh with vrious onentrtions of ethylenediminetetreti id (EDTA), Bteril isolte Con. Ainetoter Aeromons Cmpyloter Enteroous Esherihi Listeri Pseudomons Slmonell Stphyloous (mm) loetius hydrophil jejuni felis oli monoytogenes fluoresens typhimurium simulns. ± ±.84.9 ± ± ± ±.9.3 ±.8 Solution ph djusted to. with itri id. Vlues re verges ± stndrd devition. n =. -e Within olumns, different letters indite signifint (p<.) differenes. Con. = Conentrtion Tle : Size (mm) of zones of inhiition of teril isoltes y pryli id-koh with vrious onentrtions of ethylenediminetetreti id (EDTA), Bteril isolte Con. Ainetoter Aeromons Cmpyloter Enteroous Esherihi Listeri Pseudomons Slmonell Stphyloous (mm) loetius hydrophil jejuni felis oli monoytogenes fluoresens typhimurium simulns.4 ± ± ± ± ± ±.4.67 ±.99.4 ± ± ±.36. ±.4.34 ±..9 ±..33 ± ±.98.9 ± ± ±.. ± ± ±.4.4 ±.4.4 ±. Solution ph djusted to. with itri id. Vlues re verges ± stndrd devition. n =. -e Within olumns, different letters indite signifint (p<.) differenes. Con. = Conentrtion Tle 3: Size (mm) of zones of inhiition of teril isoltes produed y pri id-koh with vrious onentrtions of ethylenediminetetreti id (EDTA), Bteril isolte Con. Ainetoter Aeromons Cmpyloter Enteroous Esherihi Listeri Pseudomons Slmonell Stphyloous (mm) loetius hydrophil jejuni felis oli monoytogenes fluoresens typhimurium simulns.6 ± ± ±.7 4. ±.78.9 ± ±.9.37 ± ± ±.4.3 ± ±.4 4. ±..73 ±.47.6 ± ±..7 ± ± ± ±.6 9. ±.39.3 ± ±.63.8 ± ±.4.44 ± ± ±. Solution ph djusted to. with itri id. Vlues re verges ± stndrd devition. n =. -e Within olumns, different letters indite signifint (p<.) differenes. Con. = Conentrtion Tle 4: Size (mm) of zones of inhiition of teril isoltes produed y luri id-koh with vrious onentrtions of ethylenediminetetreti id (EDTA), Bteril isolte Con. Ainetoter Aeromons Cmpyloter Enteroous Esherihi Listeri Pseudomons Slmonell Stphyloous (mm) loetius hydrophil jejuni felis oli monoytogenes fluoresens typhimurium simulns 8.8 ±.4.8 ± ± ±.33.6 ± ± ± ± ± ±.8.97 ± ±..4 ±.3. ± ±. 9.9 ± ± ± ± ±. 8.8 ±..98 ±.37.6 ± ± ± ± ±.4 Solution ph djusted to. with itri id. Vlues re verges ± stndrd devition. n =. -e Within olumns, different letters indite signifint (p<.) differenes. Con. = Conentrtion

4 Int. J. Poult. Si., (x): xx-xx, Zones of inhiition of L. monoytogenes nd S. simulns formed y pri-koh with mm EDTA were signifintly lrger thn zones produed y the pri-koh mixtures, while inresing the EDTA onentrtion of the pri- KOH to mm produed signifint inreses in the size of the zones of inhiition of A. loetius, C. jejuni nd Slmonell typhimurium, s well s further inreses in the size of the zone of inhiition of E. felis. Also, mixtures of luri-koh supplemented with mm EDTA produed signifintly lrger zones of inhiition of A. loetius, C. jejuni, E. felis, P. fluoresens nd Slmonell typhimurium thn luri- KOH not supplemented with the heltor. Mixtures of luri-koh supplemented with mm of EDTA formed inhiition zones of A. hydrophil, L. monoytogenes nd S. simulns tht were signifintly lrger thn zones produed y luri-koh in ddition to further inreses in the size of zones of inhiition of C. jejuni nd E. felis produed y luri-koh with mm of EDTA. EDTA possesses ntiteril tivity ginst Grm negtive nd Grm positive teri (Chew et l., 98) nd the heltor hs een shown to inrese the teriidl tivity of ntimiroils (Vr, 99) euse of the ility of the heltor to inrese the permeility of ellulr memrnes (Lieve, 968; Leive, 96; Russell nd Furr, 977). Furthermore, strong heltors my inhiit miroil metolism y inding tre minerls required for ellulr reprodution, growth nd survivl (Boziris nd Adms, 999). Cheltors re routinely dded to surftnt formultions to serve s detergent uilders (Kovh, 7). These detergent uilders derese wter hrdness y inding minerls dissolved in wter, therey inresing the ility of the surftnts to remove dirt nd deris during wshing opertions. Formultions of lkline slts of ftty ids re urrently eing exmined s miroiidl surftnts tht n potentilly e used s snitizers in poultry proessing opertions (Hinton et l., 9). Alkline slts of proi, pryli, pri, luri nd myristi ids hve exhiited ntiteril tivity towrds severl teri ssoited with poultry proessing (Hinton nd Ingrm, ). The ddition of heltors to these formultions my inrese the ility of these snitizers to redue ontmintion of roiler rsses y pthogeni, spoilge nd inditor miroorgnisms. Formultions of these ntimiroil surftnts my serve s the sis for the development of more effetive poultry proessing snitizers. REFERENCES Asell, M.A. nd R.G. Egon, 966. The role of multivlent tions in the orgniztion nd struture of teril ell wlls. Biohem. Biophs. Res. Commun., : Boziris, I.S. nd M.R. Adms, 999. Effet of heltors nd nisin produed in situ on inhiition nd intivtion of grm-negtives. Int. J. Food Miroiol., 3: -3. Bulio, M. de los Angeles, R. Cngemi, M. Ceili nd G. Riden, 6. In vitro ntiteril effet of different irrigting solutions on Enteroous felis. At. Odontol. Ltinom., 9: 7-8. Chew, B.P., L.W. Tjoelker nd T.S. Tnk, 98. In vitro growth inhiition of mstitis using teri y phenolis nd metl heltors. J. Diry Si., 68: Gry, G.W. nd S.G. Wilkinson, 96. The effet of ethylenediminetetr-eti id on the ell wlls of some Grm-negtive teri. J. Gen. Miroiol., 39: Hinton, A. Jr., J.A. Cson, R.J. Buhr nd K. Liljejelke, 9. Bteri reovered from whole-rss rinstes of roiler rsses wshed in spry inet with luri id-potssium hydroxide. Int. J. Poult. Si., 8: -7. Hinton, A. Jr. nd K.D. Ingrm,. Use of the gr diffusion ssy to evlute teriidl tivity of formultions of lkline slts of ftty ids ginst teri ssoited with poultry proessing. J. Food Sfety. In press. Hinton, A. Jr. nd K.D. Ingrm,. Comprison of the ntiteril tivity of helting gents using the gr diffusion method. Int. J. Poult. Si., 9: 3-6. Kr, J.J., R. Vrle nd M.S.F. Lie Ken Jie, 977. Antimiroil lipids: Nturl nd syntheti ftty ids nd monoglyerides. Lipids, : Kovh, S.M., 7. Improve your lening proess. How wter hrdness ffets lening. Mter. Mng. Helth Cre, 6: -3. Lieve, L., 968. Studies on the permeility hnge produed in oliform teri y ethylenediminetetrette. J. Biol. Chem., 43: Leive, L., 96. Relese of lipopolyshride y EDTA tretment of E. oli. Biohem. Biophys. Res. Commun., : Ngrjn, M.K. nd H.L. Pine, 984. Wter hrdness ontrol y detergent uilders. JAOCS, 6: 47- ACKNOWLEDGMENTS The uthors knowledge the tehnil ssistne of 478. Oyrzl, O.A.,. Redution of Cmpyloter sp. Niole Brtenfeld nd Ashley Y. Willims nd the y ommeril ntimiroils pplied during the ssistne of Dr. Mihel Musgrove of the Russell proessing of roiler hikens: A review from the Reserh Center for providing the Aeromons United Sttes Perspetive. J. Food Prot., 68: 7- hydrophil isolte

5 Int. J. Poult. Si., (x): xx-xx, Phillips, C.A. nd J. Duggn,. The effet of EDTA Vr, M., 99. Agents tht inrese the permeility of nd trisodium phosphte, lone nd in omintion the outer memrne. Miroiol. Rev., 6: with nisin, on the growth of Aroter utzleri in Wilkinson, S.G., 967. The sensitivity of pseudomonds ulture. Food Miroiol., 8: to ethylene-diminetetr-eti id. J. Gen. Russell, A.D. nd J.R. Furr, 977. The ntiteril Miroiol., 47: tivity of new hloroxylenol preprtion ontining ethylenedimine tetreti id. J. Appl. Bteriol., 43: 3-6. Mention of trde nme, proprietry produt, or speifi equipment does not onstitute gurntee or wrrnty y the USDA nd does not imply its pprovl to the exlusion of other produts tht my e suitle. 4