Production of Ovotransferrin from Egg White for Antimicrobial Application. Introduction

Transcription

1 Production of Ovotrnsferrin from Egg White for Antimicrobil Appliction D. U. Ahn 1, E. J. Lee 1 nd Aubrey Mendonc 2 1 Animl Science Deprtment, Iow Stte University, Ames, Iow Ph: , Fx: , e-mil: duhn@istte.edu 2 Food Science nd Humn Nutrition, Iow Stte University, Ames, Iow Ph) , e-mil: mendon@istte.edu Introduction Ovotrnsferrin is mjor egg white protein tht cn be used s nturl ntimicrobil gent. Although, the ntimicrobil ctivity of ovotrnsferrin ginst vriety of microorgnisms hs been discussed, no ttempt hs been mde to seprte ovotrnsferrin in lrge scle nd use it s nturl ntimicrobil gent in foods. One of the mjor issues for the sustinble egg industry is the efficient movement of egg nd egg products through mrket chnnels nd incresing the vlue of egg. In recent yers, egg consumption in the U.S. strted to increse due to the incresed use of processed egg products, such s dried whole egg, yolk powder nd liquid eggs. However, the increse of egg consumption through the conventionl wy is limited. Therefore, developing new vlue-dded products nd diversifying the utiliztion of egg in new food or non-food products is necessry. There re two wys of diversifying nd incresing the use of egg: 1) seprting vlue-dded components from egg yolk nd white, nd 2) modifying physicochemicl properties of egg yolk nd white nd developing new products using the modified egg yolk nd white. Physicochemicl modifiction cn be ccomplished by enzymes, physicl, nd chemicl tretments nd cn bring new chrcteristics to egg yolk nd white. Seprtion of vlue-dded components from egg yolk nd white is more complicted nd costly, but cn increse the vlue of egg hundreds folds nd expnd the use of egg components to non-food products (e.g., phrmceuticl or ntimicrobil gents). The objectives of this work were to 1) develop commercil-scle seprtion method of food-grde ovotrnsferrin from egg white; nd 2) test the ntimicrobil cpbility of ctivted ovotrnsferrin in model nd met systems Mterils nd Methods Lrge-scle seprtion of ovotrnsferrin from egg white This proposed work is continution of previously funded project, in which we hd developed optiml seprtion conditions for ovotrnsferrin from egg white nd ctivtion conditions for its ntimicrobil ctivity. The conditions selected from the 1

2 lbortory-scle study were used s the strting conditions for lrge scle seprtion of ovotrnsferrin from egg white. However, some modifictions were mde due to drmtic increse in volume, rection nd hndling time, nd chnges in sturtion conditions for iron, mixing, centrifugtion, nd the responses of egg lbumin nd ovotrnsferrin to ph nd ethnol. Egg white (4,000 ml) ws seprted from the eggs nd diluted with the sme volume of distilled wter. Ovotrnsferrin ws seprted from other egg white proteins fter converting po-ovotrnsferrin to iron-sturted holo-ovotrnsferrin using FeCl 3. The ph of 2x-diluted egg white solution ws > ph 9.0 nd used without djusting ph. Becuse ddition of NHCO 3 nd NCl did not help iron binding much, they were eliminted fter the first round of lrge-scle tril. Ferric iron chloride (1.6 ml of 0.5M- FeCl 3.6 H 2 O solution per liter egg white solution) ws used throughout the lrge-scle method development. The iron-dded egg white solution ws homogenized using blender for 2 min. After stnding t room temperture for 1 hr, 4 volumes of 100% cold ethnol (43% finl ethnol, finl concentrtion) were dded nd egg white proteins. Holo-ovotrnsferrin ws seprted from the precipitted egg white proteins by centrifugtion t 3,220 x g for 40 min. The precipitnt ws re-extrcted with the sme concentrtion of ethnol nd centrifuged t 3,220 x g for 40 min. The superntnts were pooled nd filtered through 3 lyers of cheese cloth to remove floting mterils. After filtering, cold ethnol (100%) ws slowly dded to the superntnt to the finl ethnol concentrtion of 59% to precipitte iron-bound ovotrnsferrin. The precipitted holoovotrnsferrin ws collected fter centrifugtion t 3,220 x g for 20 min, dissolved in 9 volumes of distilled wter, nd then ethnol ws removed from the ovotrnsferrin solution using ultrfiltrtion. The ph of ethnol-free ovotrnsferrin solution ws djusted to ph 4.7 using 0.5 M citric cid nd iron ws removed using AG 1-X 2 resin in btch. After removing iron, the ovotrnsferrin ws freeze-dried. The yield nd purity of ovotrnsferrin were determined. Four replictions were prepred to clculte the yield nd purify of the finl preprtion. The prepred po-form of ovotrnsferrin ws used lone or in combintions with citric cid, EDTA, nd lysozyme for microbil tests. Bcteril strins Five different strins of E. coli O157:H7 (ATCC 43890, C467, FRIK 125, ATCC 43895, nd ) were used. Prior to inocultion, ech strin ws individully cultured in brin hert infusion broth (BHI) for 24 h t 35 o C nd hrvested in order to ctivte the strin. An liquot (5 ml) of ech culture solution ws trnsferred to sterilized centrifuge bottle nd centrifuged t 10,000 x g for 10 min t 4 o C. Pellet ws collected, wshed with sline, nd then re-suspended in 25 ml sline. An inocultion cocktil ws prepred by mixing equl volumes of ech cell suspension to hve pproximtely the sme popultion of five strins of E. coli O157:H7. 2

3 Vibility test The ntibcteril ctivity of ovotrnsferrin plus either citric cid, EDTA, or lysozyme ws investigted using vibility test. First, 2 ml of ovotrnsferrin solution (40 mg/ml) ws dded to 2 ml of 2x-concentrted BHI broth medi contining 100 mm NHCO 3. Stock solution of either EDTA or lysozyme ws dded to the ovotrnsferrin plus 100 mm NHCO 3 solution so tht ech solution contined 50 mm citric cid, 2 mg/ml of EDTA or 1 mg/ml lysozyme. Also, 40 μl of cell suspension contining 10 6 ~ 10 7 cells of ctively growing E. coli O157:H7 ws inoculted to the prepred solution to mke 10 4 ~ 10 5 CFU per ml in the initil solution. EDTA or lysozyme lone ws prepred s control group. After inocultion, the prepred culture solutions were incubted t 35 o C for 36 h. The number of vible cells ws nlyzed by spred plte of ech culture solution (0.1 ml) fter diluting (1:10) with 0.1% sterile peptone wter. The smples were incubted t 35 o C for 24 ~ 36 h. The number of survivors on BHI gr pltes ws counted s colony-forming units per ml (CFU/ml) of smple. Appliction of ovotrnsferrin on hm Commercil hms were purchsed from 3 locl retil stores, nd hms from ech store were used s replicte. Hms were sliced to 0.2 cm-thick pieces nd vcuum-pckged (-1 br of vcuum with 10 second dwell time) in low oxygenpermeble bgs (nylon/polyethylene, 9.3 ml O 2 /m 2 /24 h t 0 o C). Prior to inocultion, ech pckge ws septiclly opened using n lcohol-sterilized scissors. Actively growing E. coli O157:H7 or L. monocytogenes cocktil stock suspension (0.2 ml) ws inoculted septiclly on the surfce of sliced hm to mke 10 3 CFU/cm 2. After inoculted hm smples were mnully mixed for 30 s to distribute the inoculum evenly, the pckged smples were rndomly divided into 5 groups. Lysozyme or/nd EDTA were dded to OS solution. The tretments were s follow: 20 mg/ml ovotrnsferrin contining 100 mm NHCO 3 (1OS) plus 2 mg/ml EDTA (1OSE), 1OS plus 2 mg/ml EDTA nd 1 mg/ml lysozyme (1OSEL), 30 mg/ml ovotrnsferrin (2OS) plus 2 mg/ml EDTA (2OSE), nd 2OS plus 2 mg/ml EDTA nd 1 mg/ml lysozyme (2OSEL). One ml of ech ovotrnsferrin solution ws distributed on the sme surfce of hm nd spred mnully for 30 ~ 60 s to distribute the solution homogenously. Control group ws prepred by only inoculting E. coli O157:H7 or L. monocytogenes to hms. After dding ovotrnsferrin to hm slices, the smples were stored in 10 o C incubtor. Vible cells on the hms were nlyzed fter 0, 2, 4, 8, nd 12 d of storge: 30 ~ 50 ml of sterile 0.1% peptone wter solution ws dded to ech bg followed by pummeling for 1 min in stomcher t norml speed. After seril dilution of the smple with 0.1% peptone wter, 0.1 ml of diluted smple ws spred homogenously on the McConkey gr plte in duplicte. Survivors were enumerted s CFU per grm by the sme method presented bove. 3

4 Results nd Discussion The yield of ovotrnsferrin using the lrge-scle seprtion method ws 94.39% (Tble 1). The electrophoresis result of the seprted nd freeze-dried ovotrnsferrin indicted tht their purity ws lso greter thn 85% (Figure 1). The yield (94.39% vg.) nd purity of the finl product indicted tht the method developed is good enough for lrge-scle seprtion of ovotrnsferrin. There re few modifictions from the lbortory method: 1) sodium bicrbonte nd NCl ws eliminted becuse they did not help the seprtion of ovotrnsferrin from the white. 2) After dding ferric chloride, the egg white solution ws set t room temperture for t lest 60 min for the binding of iron to ovotrnsferrin. One precution is tht the solution should be centrifuged within 2 hr fter dding cold ethnol for the best results. 3) Nine volumes of distilled wter were used to dissolve ovotrnsferrin, which incresed the volume of ovotrnsferrin solution gretly. So, the volume of ovotrnsferrin solution should be reduced for the ese of subsequent opertions. The condenstion of ovotrnsferrin ws done using untrfiltrtion. Becuse the ovotrnsferrin solution still contined more thn 25% ethnol, ethnol ws lso removed t the ultrfiltrtion step. The removl of ethnol ws helpful in freeze-drying step t the end of the processing step. The lrge-scle seprtion process of ovotrnsferrin ws not much different from lbortory method, but the time involved ws extended significntly. So, cre should be tken to hold smples in cold room during the whole process to void proteins denturtion during the process. The ntibcteril ctivity of nturl po-ovotrnsferrin ginst E. coli O157:H7 nd L. monocytogenes in model systems incresed s the concentrtion of sodium bicrbonte incresed. Activtion of ovotrnsferrin with NHCO 3 (100 mm) mrkedly incresed ntibcteril ctivity of ovotrnsferrin ginst E. coli O157:H7 nd L. monocytogenes. Citric cid t 0.5% enhnced ntibcteril ctivity of po-ovotrnsferrin ginst E. coli O157:H7, but 0.5% citric cid lone lso showed strong bctericidl ctivity ginst L. monocytogenes. Addition of NHCO 3 nnulled the strong ntibcteril ctivity of ovotrnsferrin plus citric cid ginst the two pthogens (Fig. 3). The ntimicrobil ctivity of ovotrnsferrin ws gretly enhnced by cidic ph conditions. Zn-bound ovotrnsferrin showed bcteriosttic ctivity ginst L. monocytogenes, but Fe-bound ovotrnsferrin hd little or no ntibcteril ctivity ginst E. coli O157:H7 nd L. monocytogenes. According to these results obtined in this study, the iron binding cpcity of ovotrnsferrin does not seem to be the mjor cuse of ntibcteril ction of ovotrnsferrin (Fig. 4). Activted ovotrnsferrin (ovotrnsferrin plus 100 mm-nhco 3 ) or 0.5% citric cid did not show ntibcteril ctivity in commercil hms, indicting tht 4

5 there re mny limittions in using ovotrnsferrin to control pthogens in met or met products. Combintion of EDTA (1 mg/ml) with ctivted ovotrnsferrin (20 mg/ml) induced 3 ~ 4 log reduction in vible E. coli O157:H7 cells in brin hert infusion (BHI) broth medi, nd 1 mg/ml lysozyme plus ovotrnsferrin (20 mg/ml) resulted in 0.5 ~ 1.0 log reduction during 35 o C incubtion for 36 hr (Fig. 5). Contrry to the model system results using BHI broth, ovotrnsferrin plus EDTA or/nd lysozyme did not show ny ntimicrobil ctivities ginst E. coli O157:H7 nd L. monocytogenes in pork chops nd hms (Tble 3). The initil cell number in medi did not ffect the ntibcteril ctivity of ovotrnsferrin + EDTA or ovotrnsferrin + EDTA + lysozyme ginst E. coli O157:H7. This study demonstrtes tht combintions of ctivted ovotrnsferrin, lysozyme, nd EDTA hve potentil to control E. coli O157:H7. L. monocytogenes strted to grow fter 1 d of incubtion in the presence of > 2.0 mg/ml of lysozyme lone. The ctivted ovotrnsferrin combined with 2 mg/ml of lysozyme groups were bctericidl ginst L. monocytogenes, resulting in 1 log reduction from initil cell popultion. Even though ctivted ovotrnsferrin with lysozyme showed stronger ntibcteril ctivity thn ctivted ovotrnsferrin lone, lysozyme itself hd no significnt ntibcteril effect L. monocytogenes (Fig. 6). Also, EDTA itself t 1.0 nd 2.0 mg/ml ws bcteriosttic ginst 5 strins of L. monocytogenes, nd EDTA enhnced the ntibcteril ctivity of ctivted ovotrnsferrin ginst L. monocytogenes (Fig. 7). Contrry to in vitro test, ovotrnsferrin plus either lysozyme or EDTA, or both, did not show ny ntibcteril effect in commercil hms during storge t 10 C for 29 dys (Tble 4). Studies with pork chops lso showed similr results to hms. There were pprent differences in ntibcteril ctivities of ovotrnsferrin between model systems nd rel products. This study suggested tht ctivted ovotrnsferrin in combintion with either EDTA or lysozyme hd gret potentils s nturl ntimicrobil gents to control E. coli O157:H7 nd L. monocytogenes in broth (model systems). The lrge-scle seprtion method developed is simple, economicl, comptible for food use, nd pplicble for commercil preprtion of nturl ntimicrobil gent for foods. However, studies re needed to find why ovotrnsferrin nd its combintions do not show significnt ntimicrobil ctivities in met products. References Center for Disese Control nd Prevention, Foodborne illnesses. CDC Fct Book, Frost, G. E. nd Rosemberg, H The citrte-dependent iron trnsport system in Escherichi coli K-12. Biochim. Biophys. Act 330: Ibrhim, H. R Insights into the structure-function reltionship of ovlbumin, ovotrnsferrin nd lysozyme. In: Hen eggs: Their Bsic nd Applied Science. Ymmoto, T., Junej, I.R., Htt, H., nd Kim, M. (Eds). CRC Press. pp

6 Pkdmn, R. nd El Hge Chhine, J. M A mechnism for iron uptke by trnsferrin. Eur. J. Biochem. 236: Phelps, C. F. nd Antonini, E A study of kinetics of iron nd copper binding to hen ovotrnsferrin. Biochem. J. 147: Stdelmn, W. J., Cotterill, O.J Egg Science nd Technology. 3 rd Ed. Hworth Press Inc., Binghmpton, NY. Vlenti, P., Visc, P., Antonini, G., Orsi, N., Antonini, E The effect of sturtion with Zn +2 nd other metl ions on the ntibcteril ctivity of ovotrnsferrin. Med. Microbiol. Immunol. 176: Wrner, R. C Egg proteins. In The Proteins. Neurth H, Biley K, (Ed). AP, New York. 6

7 Figure 1. Electrophoresis of ovotrnsferrin prepred using lrge-scle method developed Lne # 1 Ovotrnsferrin Std from Sigm Lne # 2 Ovotrnsferrin R1 Lne # 3 Ovotrnsferrin R2 Lne # 4 Ovotrnsferrin R3 Lne # 5 Ovotrnsferrin R4 7

8 Figure 2. Schemtic digrm for the isoltion of ovotrnsferrin from egg white Dilution of egg white with 1 volume distilled wter Add FeCl 3 (1.6 ml of 0.5M FeCl 3.6H 2 O/L) Stir for 60 min, dd cold ethnol (43%, finl), nd centrifuge t 3,220 x g for 20 min Superntnt (1 st ) Precipitte Re-extrct with 43% ethnol Centrifugtion t 3,220 x g, 20 min Superntnt (2 nd ) Pool superntnts nd dd ethnol (superntnt: ethnol = 5:2, 59% finl) nd centrifuge Dissolve precipitnt with 9 vol. distilled wter Remove ethnol using ultrfiltrtion nd djust ph to 4.7 using 0.5M citric cid Add AG 1-X 2 resin to holo-ovotrnsferrin solution Stir for 1 hr nd filter through glss-sintered Buchner funnel Repet AG1-X 2 resin tretment by the sme method s bove Freeze dry the po-ovotrnsferrin solution 8

9 Figure 3. The influence of citric cid on ntibcteril ctivity of ovotrnsferrin ginst 10 5 CFU/ml of E. coli O157: H7 (A) nd 10 4 CFU/ml of L. monocytogenes (B) fter 2 dys of incubtion t 35 o C. The control ws inoculted with only E. coli O157:H7 or L. monocytogenes. Different letters on brs represent significntly different groups (p < 0.05, n = 4). A b b b 8.0 c 7.0 Log 10 CFU/ml d Control OTF 0.25% Citric cid OTF+ 0.25% Citric cid 0.5% Citric cid OTF + 0.5% Citric cid B Log 10 CFU/ml b b Control OTF 0.25% Citric cid OTF+ 0.25% Citric cid 0.5% Citric cid OTF + 0.5% Citric cid 9

10 Figure 4. Antibcteril ctivity of OTF, Fe-OTF, nd Zn-OTF ginst E. coli O157:H7 (10 4 CFU/ml, A) fter 1dy of incubtion t 35 o C nd L. monocytogenes (10 5 CFU/ml, B) fter 2 dys of incubtion t 35 o C. Control: only E. coli O157:H7 or L. monocytogenes. OTF: 20 mg/ml of po-ovotrnsferrin, OTF 50: OTF + 50 mm-nhco 3, OTF 100: OTF mm-nhco 3, Fe-OTF: Fe-bound ovotrnsferrin (20 mg/ml), Zn- OTF: Zn-bound ovotrnsferrin (20 mg/ml). Different letters on brs represent significntly different groups (p < 0.05, n = 4). A b b b Log10 CFU /ml c Control OTF OTF 50 OTF 100 Fe-OTF Zn-OTF B 10.0 Log 10 CFU /ml b bc c bc Control OTF OTF 50 OTF 100 Fe-OTF Zn-OTF 10

11 Figure 5. Antibcteril ctivity of ovotrnsferrin (20 mg/ml) contining 100 mm NHCO 3 nd lysozyme (1 mg/ml) or EDTA (2 mg/ml) ginst the growth of E. coli O157:H7 in BHI broth culture during 35 o C incubtion for 24 ~ 36 hr Log 10 CFU / ml bc b 2.0 c C L1 E2 OSL1 OSE2 OS C: control, only 10 4 CFU/ml of E.coli O157:H7 L1: 1 mg/ml Lysozyme E2: 2 mg/ml EDTA OS: 20 mg/ml ovotrnsferrin mm-nhco 3 OSL1: OS + 1 mg/ml lysozyme OSE2: OS + 2 mg/ml EDTA ~c letters lbeled on brs mens there were significntly different (p < 0.05, n=3) 11

12 Figure 6. Antibcteril ctivity of ovotrnsferrin (20 mg/ml) combined with 100 mm NHCO 3 nd/or lysozyme ginst the growth of L. monocytogenes on BHI broth culture during 35 o C incubtion for 24 ~ 36 hr Log 10 CFU / ml b b bc bcd cd d d 1 C L2 L2.5 L3 OL2 OL2.5 OL3 OS OSL2 OSL2.5 OSL3 -d Brs with different letters indicte significntly different vlues (P < 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 mm-nhco 3 OSL2: OS + 2 mg/ml lysozyme OSL2.5: OS mg/ml lysozyme OSL3: OS+ 3.0 mg/ml lysozyme 12

13 Figure 7. Antibcteril ctivity of ovotrnsferrin (20 mg/ml) combined with 100 mm NHCO 3 nd/or EDTA (1 mg/ml) ginst the growth of L. monocytogenes in BHI broth culture during 35 o C incubtion for 48 hr Log 10 CFU / ml b c c d d 1 C E1 E2 OS OSE1 OSE2 -d Brs with different letters indicte significntly different vlues (P < 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 mm NHCO 3 OSE1: OS + E1 OSE2: OS + E2 13

14 Tble 1. Yield of ovotrnsferrin using the finl seprtion method for ovotrnsferrin Repliction Egg white yield (g) yield (g) Avg. (%) R g % R g R g R g The yield of ovotrnsferrin ws clculted on the bsis of totl ovotrnsferrin content (13 mg/g white) in egg white. Egg white protein content used ws 10.5% of egg white nd ovotrnsferrin content vlue used ws 12.5% of egg white protein. 14

15 Tble 2. Influences of citric cid nd citrte on ntibcteril ctivity of ovtrnsferrin ginst 10 5 CFU/ml of E. coli O157:H7. Tretments Storge period (d) Number of vible cell ( log 10 CFU/ml) Citric cid 7.90 c ± c ± b ± c ± 0.43 OTF + Citric cid 4.71 d ± d ± c ± d ± 0.00 OTF + Citric cid +NHCO b ± b ± ± ± 0.15 OTF + N-Citrte 9.24 ± ± ± b ± 0.07 OTF + NHCO bc ± b ± ± b ± c Vlues with different letters within column with the sme storge dy re significntly different (p < 0.05, n=4) This study used 0.5% citric cid, 25 mm sodium citrte, nd 50 mm-nhco 3. OTF: Apo-ovotrnsferrin (20 mg/ml). 15

16 Tble 3. Survivors of E. coli O157:H7 in commercil hms treted with or without ovotrnsferrin solutions contined 100 mm-nhco 3 plus EDTA or/nd lysozyme during storge t 10 o C Tretment Storge period (d) Number of vible cell (log 10 CFU/ml) Control 3.7 ± 0.0 ** 3.8 ± ± ± 0.1 1OS + EDTA 3.5 ± ± ± ± 0.2 1OS + EDTA + Lyso 3.5 ± ± ± ± 0.3 2OS + EDTA 3.4 ± ± ± ± 0.2 2OS + EDTA + Lyso 3.3 ± ± ± ± 0.2 Vlues with different letters within column re significntly different (p< 0.05). n = 3 Control: Only E. coli O157:H7 inocultion. 1OS: Ovotrnsferrin (20 mg/ml) mm NHCO 3 2OS: Ovotrnsferrin (30 mg/ml) mm NHCO 3 Lyso: lysozyme (1 mg/ml) 16

17 Tble 4. Vible cells of L. monocytogenes on commercil hms treted with or without ovotrnsferrin in 100 mm NHCO 3 (OS) plus or/nd lysozyme (2 mg/ml) nd/or EDTA (1 mg/ml) during 10 o C storge Tretments Storge period (d) Number of vible cells (log 10 CFU/ml) Control 4.5 ± 0.0 * 4.6 ± ± ± ± ± ± 0.4 1OSL 4.3 ± ± ± ± ± ± ± 0.3 1OSLE 4.4 ± ± ± ± ± ± ± 0.4 2OSL 4.4 ± ± ± ± ± ± ± 0.6 2OSLE t 4.3 ± ± ± ± ± ± ± 0.2 L 4.3 ± ± ± ± ± ± ± 0.5 LE 4.4 ± ± ± ± ± ± ± 0.6 Mens within column with no common superscript differ (P < 0.05, n = 4). Control: only L. monocytogenes inoculted. * Men ± stndrd devition. 1OSL: 20 mg/ml ovotrnsferrin mm NHCO mg/ml lysozyme 1OSLE: 1OSL + 1 mg/ml EDTA, 2OSL: 30 mg/ml ovotrnsferrin mm NHCO mg/ml lysozyme 1OSLE: 2OSL + 1 mg/ml EDTA, L: 2 mg/ml lysozyme LE: 2 mg/ml lysozyme + 1 mg/ml EDTA 17