EDTA and Lysozyme Improves Antimicrobial Activities of Ovotransferrin against Escherichia coli O157:H7

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1 Animl Industry Report AS 656 ASL R EDTA nd Lysozyme Improves Antimirobil Ativities of Ovotrnsferrin ginst Esherihi oli O157:H7 Kyung Yuk Ko Iow Stte University Aubrey F. Mendon Iow Stte University Dong U. Ahn Iow Stte University, Reommended Cittion Ko, Kyung Yuk; Mendon, Aubrey F.; nd Ahn, Dong U. (2010) "EDTA nd Lysozyme Improves Antimirobil Ativities of Ovotrnsferrin ginst Esherihi oli O157:H7," Animl Industry Report: AS 656, ASL R2498. Avilble t: This Animl Produts is brought to you for free nd open ess by the Animl Siene Reserh Reports t Iow Stte University Digitl Repository. It hs been epted for inlusion in Animl Industry Report by n uthorized editor of Iow Stte University Digitl Repository. For more informtion, plese ontt digirep@istte.edu.

2 EDTA nd Lysozyme Improves Antimirobil Ativities of Ovotrnsferrin ginst Esherihi oli O157:H7 A.S. Leflet R2498 Kyung Y. Ko, grdute reserh ssistnt; Aubrey Mendon, professor, Deprtment of Food Siene nd Humn Nutrition; Dong Ahn, professor, Deprtment of Animl Siene Summry nd Implitions The im of this study ws to evlute the effet of ethylenediminetetreti id (EDTA) or/nd lysozyme on the ntibteril tivity of ovotrnsferrin ginst E. oli O157:H7. Ovotrnsferrin solution (20 mg/ml) ontining 100 mm-nhco 3 (OS) ws dded with EDTA (2.0 or 2.5 mg/ml), lysozyme (1.0, 1.5, or 2.0 mg/ml) or both were prepred. Antibteril tivities of OS (20 mg/ml ovotrnsferrin mm-nhco 3 ), OSE (OS+ EDTA), or OSL (OS + lysozyme) ginst E. oli O157:H7 in model systems were investigted by turbidity nd vibility tests. Also, OSE, OSL or OSEL (OS + EDTA + lysozyme) ws pplied on irrdited pork hops nd ommeril hms to determine if the solutions hve ntibteril tivity on met produts. The effet of initil ell popultion on the ntibteril tivity of ovotrnsferrin nd EDTA or lysozyme ombintions ws lso determined. EDTA t 2 mg/ml plus OS (OSE2) indued 3 ~ 4 log redution in vible E. oli O157:H7 ells in brin hert infusion (BHI) broth medi, nd 1 mg/ml lysozyme plus OS (OSL1) resulted in 0.5 ~ 1.0 log redution during 35 o C inubtion for 36 hr. However, OSE or OSEL did not show signifint ntibteril effet on pork hops nd hms during storge t 10 o C. The initil ell number in medi did not ffet the ntibteril tivity of OSE or OSEL ginst E. oli O157:H7. This study demonstrtes tht ombintions of ovotrnsferrin, NHCO 3, nd EDTA (OSE) hve potentil to ontrol E. oli O157:H7. Introdution Nturl ntimirobils tht re environmentlly friendly, medilly eptble, nd highly effetive nd eonomil to mnufture hve gined ttention due to onsumer desires for nturl food produts. Ovotrnsferrin is mjor ontributor to the egg s defense ginst mirobil infetion nd rotting. Initilly, the iron binding pbility of ovotrnsferrin, whih limits the vilbility of iron required for mirobil growth, ws onsidered s the mjor ntibteril mehnism of ovotrnsferrin. However, subsequent studies hve suggested tht the ntimirobil tion of ovotrnsferrin ould be intimtely relted to diret intertions of ovotrnsferrin with bteril surfe membrne, whih result in dmge of outer membrnes of miroorgnisms or destroy mirobil funtion suh s proton motive fore. E. oli O157:H7 possesses speifi ell-surfe ppendges tht filitte their dhesion to host-tissuemtrix omponents so tht it n tightly tth to beef tissues. In ddition, illness used by E. oli O157:H7 re frequently linked to ttle nd their produts suh s underooked ground beef nd rw milk. The ombintions of ntimirobils with different mehnisms n inrese the ntibteril effetiveness. Ethylenediminetetreti id (EDTA) hs been used in number of food produts s helting gent to prevent oxidtion nd deteriortive retions, whih re tlyzed by metl ions. Also, EDTA is known to enhne the effetiveness of ntimirobils nd ntibiotis, espeilly ginst grm-negtive bteri. EDTA destbilizes outer membrne of grm-negtive bteri by helting C 2+ nd Mg 2+ slts tht funtion s bridges between lipopolyshrides (LPS) of mirobil outer membrne, nd thus results in the relese of LPS from grm-negtive bteri. Lysozyme tlyzes the hydrolysis of 1,4-glyosidi linkges between N-etylmurmi id nd N- etylgluosmine in ell wll peptidoglyn. As the ell wlls of grm-negtive bteri re proteted by n outer membrne, grm-negtive miroorgnisms re reltively resistnt to ntimirobil tivity of lysozyme, thus the pplition of lysozyme in foods hs been limited. However, lysozyme gined onsiderble interest for use in food systems beuse it is nturl enzyme produed by mny nimls nd its tivity trgets speifi ellulr struture of miroorgnism. The im of the present study ws to promote the ntimirobil tivity of OS ginst E. oli O157:H7 through ombining EDTA nd/or lysozyme nd to identify the possibility bout pplition of the ombintions, whih re omprised of EDTA or/nd lysozyme, ovotrnsferrin, nd 100 mm-nhco 3, on ommeril hms nd irrdited pork hops to ontrol the growth of E. oli O157:H7. Mteril nd Methods Apo-ovotrnsferrin (iron-free) used in this study ws prepred by the method of Ko nd Ahn (2008). Five different strins of E. oli O157:H7 (ATCC 43890, C467, FRIK 125, ATCC 43895, nd ) were used. The ntimirobil pities of ovotrnsferrin solutions ombined with EDTA or lysozyme ginst E. oli O157:H7 were determined by mesuring the turbidity. The ntibteril tivity of ovotrnsferrin plus either EDTA or lysozyme ws investigted using vibility test.

3 Fresh boneless pork loin hops were purhsed, slied, vuum-pkged in low oxygen-permeble bgs, nd irrdited t 5 kgy to kill bteri, whih my exist in the pork hop. Eh pkge ws septilly opened nd inoulted 10 6 CFU/ ml of E. oli O157:H7 oktil stok suspension (0.2 ml) on the surfe of eh pork hop. Commeril hms were purhsed, slied nd inoulted septilly on the surfe of slied hm to mke 10 3 CFU/m 2. The number of surviving bteri ws enumerted using the sme method s in the pork hop study fter inubting for 0, 3, 8, nd 13 dys. Results nd Disussion E. oli O157:H7 in BHI broth multiplied rpidly fter 6 hr inubtion nd entered the sttionry phse fter 10 hr inubtion t 35 o C. E. oli O157:H7 in the medi ontining 2 mg/ml or 2.5 mg/ml EDTA lone showed lg time for 10 hr nd entered sttionry phse fter 18 hr of inubtion (Figure. 1), inditing tht E. oli O157:H7 strins to hve resistne to 2 mg/ml or 2.5 mg/ml EDTA. OS, OSE2 (OS + 2 mg/ml EDTA) nd OSE2.5 (OS mg/ml EDTA) tretments hd 0.1 of optil density t 620 nm (Figure. 1) inditing tht these tretments inhibited the growth of E. oli O157:H7 during 35 o C inubtion. The number of survivors in 2 mg/ml EDTA lone ws similr to tht of ontrol. However, when 2 mg/ml EDTA ws dded to OS, the ntibteril tivity of OS ws found to inrese signifintly, inditing to be bteriidl ginst E. oli O157:H7 (Figure. 3). In onlusion, even though OS ws bteriostti ginst E. oli O157:H7, the ombintion omprised of ovotrnsferrin, NHCO 3, nd EDTA (OSE2) resulted in 3 ~ 4 log redution of E. oli O157:H7. Therefore, OSE2 hs potentil s n ntibteril gent for ontrolling E. oli O157:H7. In turbidity test, lysozyme itself t 1.0, 1.5, nd 2.0 mg/ml ould not inhibit the growth of E. oli O157:H7. When both 1 mg/ml or 2 mg/ml lysozyme were ombined with OS, however, the ombintions (OSL group) hd ntibteril tivity ginst E. oli O157:H7 (Figure 2). Even though the ombintion (OSL1= OS + 1mg/ml of lysozyme) ws bteriostti ginst E. oli O157:H7, inditing tht the ntimirobil tivity of OSL1 ws due to OS. Therefore, 1 mg/ml of lysozyme did not ffet the ntimirobil tivity of OS ginst E. oli O157:H7 signifintly. It n be ssumed tht use of severl strins, s in this study, might redue the lysozyme effet on ntibteril tivity of ovotrnsferrin by induing rpid repiring of some strins. After 24 h inubtion t 35 o C, OS did not restrin ompletely the growth of E. oli O157:H7. Also, the ntibteril tion of OS ginst E. oli O157:H7 indited to be more effetive in 10 4 CFU/ml of initil ell number thn 10 6 CFU/ml. However, ntibteril tivities of OSE nd OSEL, whih were bteriidl ginst E. oli O157:H7, were not ffeted by the number of initil bteri (Figure. 4). In ddition, ombintions (EL) onsisting of EDTA (2 mg/ml) nd lysozyme (1 mg/ml) ould not inhibit the growth of E. oli O157:H7 in BHI broth medi when inoulted with 10 5 CFU/ml nd 10 6 CFU/ml ells initilly. When EL ws ombined with OS (OSEL), the solution exhibited strong ntibteriidl tivity ginst E. oli O157:H7 in BHI broth by redution round 1 log CFU /ml. E. oli O157:H7 inoulted t 10 4 CFU/m 2 grew on the pork hop nd inresed to 10 7 CFU/ml fter 10 o C storge for 12 dys. Also, 20 mg/ml OS plus 2 mg/ml EDTA (1OSE), 20 mg/ml OS ombined with 2 mg/ml EDTA nd 1 mg/ml lysozyme (1OSEL), 30 mg/ml OS plus 2 mg/ml EDTA (2OSE) or 30 mg/ml OS ombined with 2 mg/ml EDTA nd 1 mg/ml lysozyme (2OSEL) did not show ny ntimirobil tivities (Tble 1). E. oli O157:H7 grew better in pork hops, whih ontin more drip by defrosting thn ommeril hms, wheres vible E. oli O157:H7 ells inoulted on hms seemed to derese slowly unlike the pork hops. Also, 1OSE, 1OSEL, 2OSE or 2OSEL treted on hms did not show ny ntibteril tivity ginst E. oli O157:H7 nd there ws no signifint differene in the number of survivors between ontrol nd other tretments in both pork hops nd ommeril hms (p < 0.05) (Tble 2). In ontrst to the in vitro tests, lk of ntibteril tivity of ovotrnsferrin in pork hops nd ommeril hms my be due minly to divlent ions suh s C 2+ nd Mg 2+, whih re ssumed to exist on hms nd pork hops. The results obtined from pplition of ntimirobils to foods or produts re found to be signifintly different from those of the model system. Generlly, food produts provide nutrient-rih environment so tht miroorgnisms injured by ertin ntimirobils re onsidered to be reovered more esily in the systems thn under ell strvtion onditions or lbortory medi. Also, distribution or dilution effet of ovotrnsferrin on the produts ould be nother explntion for the low ntibteril tivity of ovotrnsferrin in pork hops nd hms. Even though in the present study 20 ~ 30 mg/ml of ovotrnsferrin ws distributed on the surfe of pork hops nd hms, the onentrtion ws likely to be diluted on the surfe of the produts. In ddition, signifint mount of drip formed during the defrosting proess in pork hops might prevent the omplete inorportion of ovotrnsferrin on the surfe of the pork hops. Therefore, this study demonstrtes tht ntimirobil tion by ovotrnsferrin in pork hop nd hms seemed to be modulted due to ssumed resons desribed bove. And, it is neessry to overome some limits nd pprohes to vrious study required for pplition of ovotrnsferrin on met or met produts.

4 Tble 1. Chnges of the number of E. oli O157:H7 survivors on e-bem irrdited pork hops treted with or without ovotrnsferrin solutions ontined 100 mm NHCO 3 plus either EDTA (2 mg/ml) or/nd lysozyme (1 mg/ml) during 10 o C storge. Number of vible ells (log 10 CFU/ml) Tretments Storge period (dys) Control 4.1 ± ± ± ± ± 0.1 1OSE 4.1 ± ± ± ± ± 0.7 1OSEL 4.2 ± ± ± ± ± 1.3 2OSE 4.2 ± ± ± ± ± 1.0 2OSEL 4.0 ± ± ± ± ± 1.3 Vlues with different supersripts within olumn re signifintly different (p< 0.05). n = ; Control: Only E. oli O157:H7; 1OS or 2OS: Ovotrnsferrin (20 mg/ml =1 or 30 mg/ml = 2) mm-nhco ; 1OSE: 1OS + 2 mg/ml EDT; 1OSEL: 1OS + 2 mg/ml EDTA + 1 mg/ml lysozym; 2OSE: 2OS + 2 mg/ml EDT; 2OSEL: 2OS + 2 mg/ml EDTA + 1 mg/ml lysozyme Tble 2. Survivors of E. oli O157:H7 in ommeril hms treted with or without ovotrnsferrin solutions ontined 100 mm-nhco 3 plus EDTA or/nd lysozyme during storge t 10 o C. Number of vible ells (log 10 CFU/g) Tretment Storge period (dys) Control 3.7 ± ± ± ± 0.1 1OSE 3.5 ± ± ± ± 0.2 1OSEL 3.5 ± ± ± ± 0.3 2OSE 3.4 ± ± ± ± 0.2 2OSEL 3.3 ± ± ± ± 0.2 Vlues with different supersripts within olumn re signifintly different (p< 0.05). n = 3 Control: Only E. oli O157:H7; 1OS or 2OS: Ovotrnsferrin (20 mg/ml =1 or 30 mg/ml = 2) mm-nhco ; 1OSE: 1OS + 2 mg/ml EDT; 1OSEL: 1OS + 2 mg/ml EDTA + 1 mg/ml lysozyme; 2OSE: 2OS + 2 mg/ml EDTA; 2OSEL: 2OS + 2 mg/ml EDTA + 1 mg/ml lysozyme

5 Figure 1. Turbidity of BHI broth ultures inoulted E. oli O157:H7 nd ovotrnsferrin (20 mg/ml) solution ombined NHCO 3 or/nd EDTA during 35 o C inubtion Figure 3. Antibteril tivity of ovotrnsferrin (20 mg/ml) ontining 100 mm NHCO 3 nd lysozyme (1 mg/ml) or EDTA (2 mg/ml) ginst the growth of E. oli O157:H7 in BHI broth ulture during 35 o C inubtion for 36 h. Turbidity (t 620nm) Time (hr) Log 10 CFU / ml b b E2 E2.5 OSE2 OSE2.5 C OS C L1 E2 OSL1 OSE2 OS C: ontrol, only 10 4 CFU/ml of E. oli O157:H7; E2: 2 mg/ml EDT; E2.5: 2.5 mg/ml EDT; OS: 20 mg/ml ovotrnsferrin mm-nhco ; OSE2: OS + 2 mg/ml EDT; OSE2.5: OS mg/ml EDTA. Figure 2. Turbidity of BHI broth ultures inoulted E. oli O157:H7 nd ovotrnsferrin (20 mg/ml) ombining NHCO 3 or/nd lysozyme during 35 o C inubtion. Turbidity (t 620nm) Time (hr) L1 L1.5 L2 OSL1 OSL1.5 OSL2 OS C C: ontrol, only 10 4 CFU/ml of E. oli O157:H7; L1: 1 mg/ml Lysozyme; L1.5: 1.5 mg/ml Lysozyme; L2: 2 mg/ml lysozyme; OS: 20 mg/ml ovotrnsferrin mm NHCO 3 ; OSL1: OS + 1 mg/ml lysozyme; OSL1.5: OS mg/ml lysozym; OSL2: OS + 2 mg/ml lysozyme - Different letters shows tht the mens re signifintly different (P < 0.05). n=3. C: ontrol, only 10 4 CFU/ml of E. oli O157:H7; L1: 1 mg/ml Lysozyme; E2: 2 mg/ml EDTA OS: 20 mg/ml ovotrnsferrin mm-nhco ; OSL1: OS + 1 mg/ml lysozyme OSE2: OS + 2 mg/ml EDTA Figure 4. Effet of initil ell popultion on ntibteril tivity of ovotrnsferrin (20 mg/ml) ombining 100 mm NHCO 3 nd EDTA (2 mg/ml) or Lysozyme (1 mg/ml) ginst E. oli O157:H7 in BHI broth ulture during 35 o C inubtion for 24 h. Log 10 CFU /ml b b 1 OS (104) OS (105) OS (106) OSE (104) OSE (105) OSE (106) OSEL (104) OSEL (105) OSEL (106) EL (105) EL (106) - Different letters shows tht the mens re signifintly different (P < 0.05). n= : 10 4 CFU/ ml BHI broth; 105: 10 5 CFU/ ml BHI broth; 106: 10 6 CFU/ ml BHI broth ; OS: 20 mg/ml ovotrnsferrin mm-nhco 3 ; OSE: OS + 2 mg/ml EDTA; OSEL: OS + 2 mg/ml of EDTA + 1 mg/ml lysozyme; EL: 2 mg/ml EDTA + 1 mg/ml lysozyme