Animl Inustry Report AS 656 ASL R2495 2010 Effect of EDTA n Lysozyme on the Antimicroil Activity of Ovotrnsferrin ginst Listeri monocytogenes Kyung Yuk Ko Iow Stte University Aurey F. Menonc Iow Stte University Dong U. Ahn Iow Stte University, uhn@istte.eu Recommene Cittion Ko, Kyung Yuk; Menonc, Aurey F.; n Ahn, Dong U. (2010) "Effect of EDTA n Lysozyme on the Antimicroil Activity of Ovotrnsferrin ginst Listeri monocytogenes," Animl Inustry Report: AS 656, ASL R2495. Aville t: https://li.r.istte.eu/ns_ir/vol656/iss1/16 This Animl Proucts is rought to you for free n open ccess y the Animl Science Reserch Reports t Iow Stte University Digitl Repository. It hs een ccepte for inclusion in Animl Inustry Report y n uthorize eitor of Iow Stte University Digitl Repository. For more informtion, plese contct igirep@istte.eu.
Effect of EDTA n Lysozyme on the Antimicroil Activity of Ovotrnsferrin ginst Listeri monocytogenes A.S. Leflet R2495 Kyung Y. Ko, grute reserch ssistnt; Aurey Menonc, professor, Deprtment of Foo Science n Humn Nutrition Dong Ahn, professor, Deprtment of Animl Science Summry n Implictions This stuy evlute the effect of EDTA n lysozyme on the nticteril ctivities of ctivte ovotrnsferrin ginst 5 strins of L. monocytogenes. First, isc test ws performe to screen the concentrtions of EDTA or lysozyme tht showe nticteril ctivities in ovotrnsferrin (O) or ovotrnsferrin in 100 mm NHCO 3 (OS) solution. Turiity n viility test were conucte using O or OS solution comine with either lysozyme (OL n OSL) or EDTA (OE n OSE). Also, OS comine with 2 mg/ml lysozyme (OSL) or/n 1 mg/ml EDTA (OSLE) were pplie on commercil hms to etermine if the solutions show nticteril ctivities on met proucts. The effect of initil cell popultion on the nticteril ctivities of ovotrnsferrin comine with either EDTA or lysozyme ws lso etermine. L. monocytogenes strte to grow fter 1 y of incution in the presence of > 2.0 mg/ml lysozyme. OL groups showe wek nticteril ctivities ginst L. monocytogenes in BHI roth culture n their ctivities were cteriosttic. OSL groups were ctericil ginst L. monocytogenes, resulting in 1 log reuction from initil cell popultion. Even though OSL showe stronger nticteril ctivity thn OS, lysozyme h no significnt effect on nticteril ctivity of OS ginst L. monocytogenes. Also, EDTA itself t 1.0 n 2.0 mg/ml were cteriosttic ginst 5 strins of L. monocytogenes. They were more susceptile to EDTA thn lysozyme, n OSE1 n OSE2 h ctericil ctivity ginst L. monocytogenes. There ws significnt ifference in the survivor cell popultions etween OS n OSE groups (p < 0.05). Therefore, EDTA enhnce the nticteril ctivity of OS ginst L. monocytogenes. However, ovotrnsferrin plus either lysozyme or/n EDTA i not show ny nticteril effect in commercil hms uring storge t 10 o C. In ition, the initil popultion of L. monocytogenes cells influence the nticteril ctivity of OSL or OSE. Introuction Nturl ntimicroil gents originte from plnt, niml, n cteril sources re expecte to provie gret stisfction to consumers who re sensitive to helth n sfety issues. Ovotrnsferrin, the secon mjor egg white protein, is n iron-ining glycoprotein n trnsports n scvenges Fe (III) in eggs of poultry. Anticteril properties of ovotrnsferrin ginst vriety of microorgnisms hve een reporte. However, the ntimicroil mechnisms of ovotrnsferrin re not fully efine yet. The iron ining cpility of ovotrnsferrin ws initilly elieve to e the mjor ntimicroil ction of ovotrnsferrin. However, recent stuies emonstrte tht irect interctions of ovotrnsferrin with cteril surfce were the mjor cuse of ntimicroil ction of ovotrnsferrin. L. monocytogenes cn proliferte in the presence of curing slt t refrigerte tempertures, n colonize, multiply n persist on processing equipment or plnt sustnces. The strins re sensitive to het tretment n cn e esily inctivte y cooking. However, rey-toet (RTE) met proucts such s frnkfurters, n eli mets hve high incience rtes of listeriosis ue to contmintion uring multiple hnling n processing steps performe fter cooking. Center for Disese Control n Prevention reporte tht 2,500 people suffer from listeriosis nnully in the Unite Sttes n one in five ie from the isese (FSIS). Due to this risk, the U.S. Deprtment of Agriculture currently estlishe zero tolernce policy for L. monocytogenes in RTE met proucts. Therefore, eveloping potent ntimicroils ginst L. monocytogenes for RTE met is necessry. Accoring to previous stuy, po-ovotrnsferrin lone ws not effective in controlling L. monocytogenes in BHI roth. To improve the ntimicroil ctivities of ovotrnsferrin, therefore, comintions of severl ntimicroil gents re necessry. The comintions of mterils with ifferent ntimicroil mechnisms re expecte to increse nticteril effectiveness since simultneous ttck on ifferent trgets is likely to mke the microorgnism more ifficult to overcome the environment. The use of ovotrnsferrin comine with EDTA or/n lysozyme s ntimicroils on the surfce of met or met proucts is ppeling to the met inustry ecuse ll the components re generlly regre s sfe (GRAS). Lysozyme ctlyzes the hyrolysis of (1-4) glycosiic linkges etween N-cetylmurmic ci n N-cetylglucosmine of cell wll peptioglycn. Therefore, the ntimicroil ctivity of lysozyme is scrie primrily to the enzymtic lyses of peptioglycn in the cell wll of microorgnisms. The ppliction of lysozyme in foos hs een limite ecuse grm-negtive cteri tht re protecte y n outer memrne, which is reltively resistnt to ntimicroil ctivities of lysozyme. However, lysozyme gine consierle interest for use in foo systems ecuse it is nturl enzyme prouce y nimls n its ctivity trgets specific cellulr structure of microorgnisms. Ethyleneiminetetrcetic ci (EDTA) hs een use in numer of foo proucts s chelting gent to
prevent oxition. Although the mechnism of nticteril effectiveness y EDTA is not fully unerstoo, its chelting property to ivlent ctions like C 2+ or Mg 2+ shoul e involve. EDTA inhiits the growth of microorgnisms y epriving Mg 2+, C 2+, n Fe 2+, which re essentil fctors for microil growth, from microorgnisms. EDTA is reporte to enhnce the ntimicroil ctivity of lctoferrin y estilizing the outer memrne of cteri n incresing the permeility of ivlent ctions. Also, chelting gents fcilitte the etchment of cells from iofilm n enhnce the killing of iofilm-proucing microorgnisms y epriving Mg 2+ ssocite with lipopolyscchries. The ojective of this stuy ws to enhnce the ntimicroil ctivity of ovotrnsferrin solution supplemente with 100 mm-soium icronte ginst cocktil of 5 strins of L. monocytogenes y comining with lysozyme or EDTA. Mny stuies relte to nticteril ctivity of ovotrnsferrin in vitro hve een reporte, ut little work hs een one to pply ovotrnsferrin s n ntimicroil gent in met or met proucts. Therefore, the present stuy pplie OS plus lysozyme or/n EDTA on commercil hms to etermine their effect on the growth of L. monocytogenes in met proucts. Mteril n Methos Apo-ovotrnsferrin (iron-free) use in this stuy ws prepre y the metho of Ko n Ahn. Five ifferent strins of Lister monocytogenes (NADC 2045, H7962, H7969, H7762, n H7596) were use. Prior to nlyzing the nticteril ctivity through viility n turiity tests, isc iffusion metho ws use to etermine the concentrtion of EDTA or lysozyme cple of promoting nticteril cpility of ovotrnsferrin. L. monocytogenes cocktil contining equl mount of ech strin ws ilute in series n then 0.1 ml of cell suspension corresponing to 10 5 or 10 6 CFU/ml ws spre homogenously on the BHI gr mei pltes. The ntimicroil cpcity of ovotrnsferrin solutions comine with EDTA or lysozyme ginst the growth of L. monocytogenes ws nlyze y mesuring the turiity of solution fter incution in BHI roth culture t 35 o C. The nticteril ctivities of ovotrnsferrin plus lysozyme (OSL) n ovotrnsferrin plus EDTA (OSE) were investigte using viility test. Commercil hms were slice, inoculte on the surfce with ctivte L. monocytogenes cocktil stock suspension n vcuum pckge in low oxygenpermele gs. After mnully mixing for 30 secon to istriute the inocul evenly, the pckge smples were rnomly ivie into 7 groups. Lysozyme or/n EDTA were e to ovotrnsferrin solution inclue 100 mm NHCO 3 n 0.15 M NCl. Ech prepre ovotrnsferrin solution (1 ml) ws istriute evenly on the surfce of hm. Vile L. monocytogenes cells on the pork chops were nlyze fter 0, 2, 5, 10, 15, 22, n 29 ys of storge. Results n Discussion Lysozyme t 1.0 n 1.5 mg/ml i not form cler zones, ut 2.0, 2.5 n 3.0 mg/ml lysozyme i. The strength of cler zone forme y lysozyme comine with ovotrnsferrin or OS i not show significnt ifference compre with tht of lysozyme (Tle 1). L. monocytogenes ws susceptile to 2.0 mg/ml lysozyme. The ntimicroil ctivity of lysozyme hs long een elieve to e ue to its cteriolytic ctions, which hyrolyze ß-1.4-glycosiic on in peptioglycn of grm-positive cteri tht o not possess ny outer memrne, ut grm-negtive cteri with outer memrne is reporte to e reltively resistnt to lysozyme. EDTA t 1.5 n 2.0 mg/ml me cler zone, ut no cler zone ws forme t 1.0 mg/ml EDTA. When EDTA ws comine with ovotrnsferrin or OS, the strength of cler zone ecrese (Tle 1). This stuy inicte tht more thn 1.5 mg/ml of EDTA inhiite the growth of L. monocytogenes. However, when EDTA ws comine with ovotrnsferrin or OS, the strength of cler zone ecrese. Bse on the isc test, more thn 2 mg/ml lysozyme or 1 mg/ml of EDTA were chosen in turiity n viility tests, n tests were performe to ientify EDTA n lysozyme effects on nticteril ctivities of ovotrnsferrin. L. monocytogenes entere log phse fter 7 ~ 8 hr of incution t 35 o C in BHI roth culture. They seeme to fce sttionry phse fter 24 hr incution n then eth. Lysozyme t 2 mg/ml elye the growth of L. monocytogenes for 24 hrs, wheres lysozyme 2.5 mg/ml n 3 mg/ml elye it for 30 hrs n 36 hr, respectively. Therefore, L. monocytogenes seeme to hve lg time to pt to new environment when lysozyme ws e (Figure 1). Even though lysozyme lone elye the growth of L. monocytogenes uring initil perio, L. monocytogenes strte to grow fter 24 ~ 36 hr incution t 35 o C. The turiity vlues otine from ll OL or OSL groups were lower thn 0.1 s in OS tretment (Figure 1). Accoring to the turiity test, L. monocytogenes were foun to e controlle y OL or OSL groups even though they were resistnt to lysozyme lone uner the concentrtions use. In the viility test, which mesures the numer of vile L. monocytogenes cells fter 48 hr incution t 35 o C, with 2.0, 2.5 or 3.0 mg/ml lysozyme ws not ifferent from tht of the control (Figure 2). As BHI mei use in this stuy is reltively nutrient-rich mei, injure cells of L. monocytogenes y lysozyme tretment shoul hve een repire esily in the mei. Thus they showe resistnce to 2.0 ~ 3.0 mg/ml lysozyme. Another possile explntion for resistnce of L. monocytogenes to lysozyme my e ttriute to using cocktil of 5 ifferent strins of L. monocytogenes inste of using specific strin. The numer of vile cells otine when using OS slightly increse from 4.6 log CFU/ml, the numer of the initil inocultion, to 4.9 log CFU/ml uring 48 hrs incution t 35 o C. Therefore, OS seeme to e
cteriosttic ginst L. monocytogenes. Also the numer of vile L. monocytogenes cells in OL2, OL2.5, n OL3 increse from 4.6 log CFU/ml, the numer of initil inoculte cell, to 5.1, 7.2, n 6.0 log CFU/ ml, respectively (Figure 2). OL groups showe wek nticteril ctivity ginst L. monocytogenes compre with control. However, OSL groups showe stronger ntilisteril ctivity ginst L. monocytogenes thn tht of OL groups. In ition, OSL2, OSL2.5, n OSL3.0 were listericil consiering their chnges from 4.6 log CFU/ml to 3.3, 3.0 or 2.9 log CFU/ml, respectively. Also there ws significnt ifference in nticteril ctivity ginst L. monocytogenes etween OL n OSL uner lysozyme t 2.5 mg/ml n 3.0 mg/ml (Figure 2). Therefore, this stuy emonstrtes tht 100 mm soium icronte enhnce the ntimicroil ctivity of OL ginst L. monocytogenes, while lysozyme t 2.0, 2.5, or 3.0 mg/ml i not show cler effect on nticteril ctivity of OS towr L. monocytogenes. L. monocytogenes were susceptile to EDTA s shown in the isc test. As EDTA lone showe lower turiity thn OE or OSE t initil point, it ws impossile to compre the nticteril ctivity of OE or OSE with EDTA itself y turiity nlysis (Figure 3). As lmost ll tretments showe similr turiity vlues of less thn 0.1, it ws ifficult to etermine if EDTA h synergistic effect with ovotrnsferrin using turiity test. However, ccoring to the turiity test, EDTA increse the nticteril ctivity of OS (Figure 3). L. monocytogenes ws foun to e more susceptile to EDTA thn lysozyme uner the concentrtions use in this stuy (Figure 4). EDTA increse the nticteril ctivity of OS ginst L. monocytogenes. However, ntimicroil effects y EDTA itself seeme to e the mjor contriutor to the listericil ctivity of OSE. OS tretment to BHI roth mei inoculte with 10 4 n 10 5 CFU/ml occurre 1 log increse from the initil cell numer uring 35 o C incution for 48 hrs. However, the vile cells in BHI roth culture inoculte with 10 6 cells n trete with OS i not inicte ny increse (Figure 5). BHI roth culture e with OSL2 n inoculte with 10 4, 10 5 or 10 6 CFU/ml of L. monocytogenes resulte in 3.3, 3.7, or 4.5 log CFU/ml of vile cells, respectively, fter 48 hrs incution t 35 o C. BHI roth inoculte with 10 4, 10 5 or 10 6 CFU/ml of L. monocytogenes cells n OSE1 showe 3.1, 4.0, or 4.4 log CFU/ml of vile cells, respectively (Figure 5). These results emonstrte tht OSL2 or OSE1 h listericil ctivity ginst L. monocytogenes n the numer of initil cells existing in the mei ffecte the nticteril ctivity of OSL2 n OSE1. L. monocytogenes with 10 4 initil cells grew slowly in commercil hms to 10 5 CFU/ml uring storge t 10 o C for 29 ys. In contrst to in vitro test, 1OSL, 1OSLE, 2OSL, n 2OSLE i not show ny ntimicroil ctivity ginst L. monocytogenes (Tle 2). Therefore, it ws suggeste tht even though OSL2 n OSE1 showe cler ctericil effect ginst L. monocytogenes in moel systems, they i not hve nticteril ctivities ginst L. monocytogenes in commercil hms uring storge. EDTA plus lysozyme i not show ny ntimicroil ctivity ginst L. monocytogenes in hms in this stuy. Such ifference my e ue prtilly to mei compositions. As foo proucts provie nutrient-rich environment, injure microorgnisms y ntimicroils my recover more esily in foo systems thn those uner cell strvtion or limite mei conitions. Another possile explntion out no nticteril ctivities y OSL n OSE in commercil hms coul e istriution prolem or ilution effect of ovotrnsferrin to the proucts, stte of strins in inocul, or stility of ovotrnsferrin on the surfce of met proucts. Generlly, ntimicroil ctivity of ovotrnsferrin is epenent upon the ose of ovotrnsferrin. In the present stuy, 1 ml of 20~ 30 mg/ml ovotrnsferrin ws istriute on the surfce of hms, n the concentrtion ws likely to e ilute in certin prts of the prouct so tht low nticteril effect ginst L. monocytogenes might e shown in the re. Also, ovotrnsferrin ws spre mnully, n thus it is ifficult to istriute ovotrnsferrin uniformly or homogenously on whole proucts. Also, ovotrnsferrin incorporte coul hve een penetrte or sore insie the prouct so tht it coul not e involve in ntimicroil ctivities. Even though ntimicroils such s cteriocins re effective in inhiiting pthogens incluing L. monocytogenes n other spoilge microorgnisms in moel systems, high popultion of survivors or recovering fetures is often oserve in foo systems. This stuy showe tht there re mny limittions in pplying ovotrnsferrin s ntimicroil gent in foo systems.
Tle 1. Effect of ovotrnsferrin comine with NHCO 3 n EDTA or NHCO 3 n lysozyme on cler zone formtion on BHI gr mei pltes inoculte with L. monocytogenes uring 35 o C incution for 24 ~ 48 hrs. Tretment Conc. (mg/ml) Control OTF OTF + NHCO 3 0 - * - -/+ 1.0 - - - 1.5 - +/- - Lysozyme 2.0 ++ ++ +/- 2.5 ++ ++ ++ 3.0 ++ +++ ++ 1.0 - - - EDTA 1.5 ++ + ** + 2.0 ++ + ++ 20 mg/ml ovotrnsferrin 100 mm NHCO 3 ws use t this stuy. * No cler zone forme on BHI gr mei plte. ** Formtion of cler zone: the strength of cler zone ws escrie s + (wek), ++ (mile), n +++ (strong) y its size or clrity on the BHI gr. (n = 3) Tle 2. Vile cells of L. monocytogenes on commercil hms trete with or without ovotrnsferrin in 100 mm NHCO 3 (OS) plus or/n lysozyme (2 mg/ml) n/or EDTA (1 mg/ml) uring 10 o C storge. Tretments Storge perio (ys) 0 2 5 10 15 22 29 ------------------------------------ Numer of vile cells (log 10 CFU/ml) ---------------------------------------- Control 4.5 ± 0.0 * 4.6 ± 0.2 5.0 ± 0.0 5.3 ± 0.4 5.8 ± 0.0 5.4 ± 0.2 5.3 ± 0.4 1OSL 4.3 ± 0.1 4.7 ± 0.1 5.1 ± 0.1 5.5 ± 0.1 5.6 ± 0.4 5.5 ± 0.9 5.2 ± 0.3 1OSLE 4.4 ± 0.1 4.6 ± 0.0 5.0 ± 0.1 5.1 ± 0.2 5.9 ± 0.3 5.6 ± 0.2 6.0 ± 0.4 2OSL 4.4 ± 0.0 4.6 ± 0.0 5.0 ± 0.1 5.2 ± 0.2 5.2 ± 0.1 5.5 ± 0.0 5.4 ± 0.6 2OSLE t 4.3 ± 0.1 4.6 ± 0.1 4.9 ± 0.2 5.1 ± 0.1 5.6 ± 0.2 5.7 ± 0.3 5.4 ± 0.2 L 4.3 ± 0.0 4.5 ± 0.1 4.9 ± 0.1 4.9 ± 0.3 5.4 ± 0.3 5.0 ± 1.0 5.4 ± 0.5 LE 4.4 ± 0.2 4.5 ± 0.1 5.0 ± 0.1 5.1 ± 0.1 5.5 ± 0.5 5.6 ± 0.3 6.2 ± 0.6 Mens within column with no common superscript iffer (P < 0.05, n = 4). Control: only L. monocytogenes inoculte. * Men ± stnr evition. 1OSL: 20 mg/ml ovotrnsferrin + 100 mm NHCO 3 + 2 mg/ml lysozyme 1OSLE: 1OSL + 1 mg/ml EDTA, 2OSL: 30 mg/ml ovotrnsferrin + 100 mm NHCO 3 + 2 mg/ml lysozyme 1OSLE: 2OSL + 1 mg/ml EDTA, L: 2 mg/ml lysozyme LE: 2 mg/ml lysozyme + 1 mg/ml EDTA
Figure 1. Turiity of BHI roth cultures inoculte L. monocytogenes n ovotrnsferrin (20 mg/ml) comine with NHCO 3 or/n EDTA uring 35 o C incution. Figure 3. Turiity of BHI roth cultures inoculte L. monocytogenes n ovotrnsferrin (20 mg/ml) comine with NHCO 3 or/n EDTA uring 35 o C incution. Turiity (t 620nm) 1.2 1 0.8 0.6 0.4 0.2 0-0.2 0 10 20 30 40 50 Time (hrs) Control L2 L2.5 L3 OS OL2 OL2.5 OL3 OSL2 OSL3 OSL2.5 Opticl ensity t 620nm 0.10 0.08 0.06 0.04 0.02 0.00-0.02-0.04 0 10 20 30 40 50 60 Time (hrs) E1 E1.5 E2 OE1 OE1.5 OE2 OSE1 OSE1.5 OSE2 OS C: control, 10 4 CFU/ml of L. monocytogenes; L2: 2 mg/ml Lysozyme; L2.5: 2.5 mg/ml Lysozyme; L3: 3.0 mg/ml Lysozyme; OS: 20 mg/ml ovotrnsferrin + 100 mm-nhco 3 ; OL2: 20 mg/ml ovotrnsferrin + L2; OL2.5: 20 mg/ml ovotrnsferrin + L 2.5; OL3: 20 mg/ml ovotrnsferrin + L 3.0; OSL2: OS + 2 mg/ml lysozyme; OSL2.5: OS + 2.5 mg/ml lysozyme; OSL3: OS+ 3.0 mg/ml lysozyme C: control, only 10 4 CFU/ml of L. monocytogenes E1: 1 mg/ml EDTA; E1.5: 1.5 mg/ml EDTA; E2: 2.0 mg/ml EDTA; OS: 20 mg/ml ovotrnsferrin + 100 mm NHCO 3 ; OE1: 20 mg/ml ovotrnsferrin + E1; OE1.5: 20 mg/ml ovotrnsferrin + E2; OE2: 20 mg/ml ovotrnsferrin + E3; OSE1: OS + E1; OSE1.5: OS + E1.5; OSE2: OS+ E2 Figure 2. Anticteril ctivity of ovotrnsferrin (20 mg/ml) comine with 100 mm NHCO 3 n/or lysozyme ginst the growth of L. monocytogenes on BHI roth culture uring 35 o C incution for 24 ~ 36 hr. Log 10 CFU / ml 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 c 1 C L2 L2.5 L3 OL2 OL2.5 OL3 OS OSL2 OSL2.5 OSL3 c c - Brs with ifferent letters inicte significntly ifferent vlues (P < 0.05. n=4). C: control, only 10 4 CFU of L. monocytogenes; L2: 2 mg/ml Lysozyme; L2.5: 2.5 mg/ml Lysozyme; L3: 3.0 mg/ml Lysozyme; OL2: 20mg/ml ovotrnsferrin + L2; OL2.5: 20mg/ml ovotrnsferrin + L2.5; OL3: 20mg/ml ovotrnsferrin + L 3.0; OS: 20 mg/ml ovotrnsferrin + 100 mm-nhco 3 ; OSL2: OS + 2 mg/ml lysozyme; OSL2.5: OS + 2.5 mg/ml lysozyme; OSL3: OS+ 3.0 mg/ml lysozyme.
Figure 4. Anticteril ctivity of ovotrnsferrin (20 mg/ml) comine with 100 mm NHCO 3 n/or EDTA (1 mg/ml) ginst the growth of L. monocytogenes in BHI roth culture uring 35 o C incution for 48 hr. Log 10 CFU / ml 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 c c 1 C E1 E2 OS OSE1 OSE2 - Brs with ifferent letters inicte significntly ifferent vlues (P < 0.05. n=4). C: control, only 10 4 cells of L. monocytogenes E1: 1 mg/ml EDTA E2: 2.0 mg/ml EDTA OS: 20 mg/ml ovotrnsferrin + 100 mm NHCO 3 OSE1: OS + E1 OSE2: OS + E2 Figure 5. Effect of initil cell popultion on the nticteril ctivity of ovotrnsferrin (20 mg/ml) solution comine with 100 mm NHCO 3 n EDTA (2 mg/ml) or lysozyme (1 mg/ml) ginst L. monocytogenes in BHI roth culture uring 35 o C incution for 48 hr. Log 10 CFU / ml 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 e e ce c OS (A) OSL2 (A) OSE1 1 (A) OS (B) OSL2 (B) OSE1 (B) OS (C) OSL2 (C) OSE1 (C) - Brs with ifferent letters inicte significntly ifferent vlues (P < 0.05. n=4). A: 10 4 CFU/ ml BHI roth B: 10 5 CFU/ ml BHI roth C: 10 6 CFU/ ml BHI roth OS: 20 mg/ml ovotrnsferrin + 100 mm NHCO 3 OSL2: OS + 2 mg/ml lysozyme OSE1: OS + 2 mg/ml lysozyme + 1 mg/ml of EDTA c