Combined Treatment with Mild Heat, Manothermosonication and Pulsed Electric Fields Reduces Microbial Growth in Milk.

Transcription

1 Dulin Institute of Tehnology Artiles Shool of Food Siene nd Environmentl Helth 2013 Comined Tretment with Mild Het, Mnothermosonition nd Pulsed Eletri Fields Redues Miroil Growth in Milk. Rhel Hlpin Dulin Institute of Tehnology, O. Cregenzán-Alerti Shool of Agriulture nd Food Siene, University College Dulin P. Whyte Shool of Veterinry Mediine, University College Dulin J.G Lyng Shool of Agriulture nd Food Siene, University College Dulin F. Noi Shool of Agriulture nd Food Siene, University College Dulin Follow this nd dditionl works t: Prt of the Biohemistry, Biophysis, nd Struturl Biology Commons, nd the Food Biotehnology Commons Reommended Cittion Hlpin, R. et l. (2013) Comined Tretment with Mild Het, Mnothermosonition nd Pulsed Eletri Fields Redues Miroil Growth in Milk. Food Control, Volume 34, Issue 2, Deemer 2013, Pges DOI : /j.foodont This Artile is rought to you for free nd open ess y the Shool of Food Siene nd Environmentl Helth t ARROW@DIT. It hs een epted for inlusion in Artiles y n uthorized dministrtor of ARROW@DIT. For more informtion, plese ontt yvonne.desmond@dit.ie, rrow.dmin@dit.ie, rin.widdis@dit.ie. This work is liensed under Cretive Commons Attriution- Nonommeril-Shre Alike 3.0 Liense

2 1 2 3 Comined tretment with mild het, mnothermosonition nd pulsed eletri fields redues miroil growth in milk. 4 Hlpin, R.M. 1*, Cregenzán-Alerti, O. 1, Whyte, P. 2, Lyng, J.G. 1 nd Noi, F Shool of Agriulture nd Food Siene, University College Dulin, Irelnd, 6 2 Shool of Veterinry Mediine, University College Dulin. Irelnd Current ddress: Hotel Shool, Glwy-Myo Institute of Tehnology, Glwy Cmpus, Dulin Rod, Glwy, Irelnd * Corresponding uthor: Dr. Rhel Hlpin. Postl ddress: Shool of Agriulture nd Food Siene, University College Dulin, Belfield, Dulin 4, Irelnd. Tel No.: E-mil: rhel.hlpin@ud.ie

3 21 Astrt In reent yers, there hs een onsiderle interest in non-therml milk proessing. The ojetive of the present study ws to ssess the effiy of two non-therml tehnologies (mnothermosonition; MTS, nd pulsed eletri fields; PEF) in omprison to therml psteuristion, y ssessing the miroiologil qulity of eh of these milk smples postproessing. Homogenised milk ws sujeted to MTS (mplitude; 27.9 µm, pressure; 225 kp) t two tempertures (37 C or 55 C), efore eing immeditely treted with PEF (eletri field strength; 32 kv/m, pulse width; 10 µs, frequeny; 320 Hz). Therml psteuristion (72 C, 20 s) ws inluded s ontrol tretment. Miroil ontent of eh milk smple ws monitored over 21-dy period. It ws determined tht milks treted with MTS/PEF t 37 C nd 55 C ontined lower miroil levels for ertin durtion, ut fter 14 dys milk whih hd een psteurised y onventionl methods ontined signifintly (P <0.05) less miroorgnisms. However, milks treted with MTS/PEF ontined signifintly (P<0.05) fewer miroorgnisms thn rw milk t eh time point. Although not s effetive s therml psteuristion, the present study demonstrtes the ility of MTS/PEF tretment to redue miroil ontent of milk, while voiding prolonged het exposure to tempertures suh s those used during onventionl (therml) psteuristion Keywords: non-therml proessing, mnothermosonition, pulsed eletri fields, milk, miroiologil qulity

4 44 1. Introdution Milk is nutritious medium whih presents n idel environment for the growth of mny pthogeni nd spoilge miroorgnisms (Pered et l., 2009). Consequently, milk hs reltively short shelf-life. Milk is usully proessed y het tretment in order to prolong its shelf-life, nd lthough these therml tretments destroy miroorgnisms, they n lso hve detrimentl effets on the nutritionl nd orgnolepti properties of milk (Odriozol-Serrno, Bendiho-Port, & Mrtin-Belloso, 2006). The demnd y onsumers for fresh, sfe foods with fvourle sensory properties while lso hving n dequte shelf-life is diffiult need for the food industry to meet while using therml tretment (Pered et l., 2009). Thus, in reent yers there hs een onsiderle interest in the non-therml proessing of foods nd everges Non-therml proessing tehnologies require signifintly less het thn therml proessing, or in some ses no het t ll, nd re onsequently less energy-intensive nd more environmentlly friendly thn therml methods (Piysen, Mohre, & MKellr, 2003). In ddition, foods/everges sujeted to non-therml proessing re regrded s eing fresh-like, with miniml loss of olour, flvour nd nutrients (Bermudez-Aguirre, Mwson, Versteeg, & Bros-Cnovs, 2009). Exmples of suh non-therml proesses inlude mirofiltrtion, pulsed-light intivtion nd high hydrostti pressure. Two nontherml proessing methods of prtiulr relevne to the present study re ultrsonition nd pulsed eletri field tehnology Ultrsound (US) is defined s sound wves with frequenies ove the threshold of humn hering (>16 khz) (Mns & Pgn, 2005), nd this tehnology n e used to dmge or disrupt miroil ells (Doltowski, Stdnik, & Stsik, 2007). During sonition, soni wve meets liquid medium nd regions of lternting ompression nd expnsion 3

5 re reted. These regions of pressure hnge use vittion, nd ules re formed in the medium. During the expnsion yle, the ules expnd, nd point is rehed where the ule implodes nd rpid ondenstion ours. The ondensed moleules ollide violently nd shok wves re reted, whih re of very high pressures nd tempertures of up to 50MP nd 5500 C, respetively (Doltowski, Stdnik, & Stsik, 2007). These resulting hnges in pressure nd hot zones n kill teri, with the former eing the min ontriutor to miroil intivtion (Piysen, Mohre, & MKellr, 2003). In order to inrese ntimiroil effiy, sonition n e omined with het (thermosonition; TS), pressure (mnosonition; MS), or het nd pressure (mnothermosonition; MTS). However, the effetiveness of ultrsound is dependent on the type of miroorgnism trgeted, the mplitude of the ultrsoni wves, tretment time nd the volume nd omposition of the food eing treted (Doltowski, Stdnik, & Stsik, 2007). With regrd to miroil suseptiility, Grm positive teri re generlly more sensitive thn Grm negtive miroorgnisms, nd spores re onsidered to e more resistnt to tretment with ultrsound thn vegettive ells (Burgos, 1998) Another non-therml intivtion method whih is gining populrity in terms of ommeril interest is pulsed eletri field (PEF) tehnology (Devlieghere, Vermeiren, & Deevere, 2004). This method is sed on pulsing power delivered to food/everge pled etween set of eletrodes within hmer. Essentilly, series of high voltge pulses re pplied etween the set of eletrodes, whih uses disruption to nd susequent permeilistion of miroil ell memrnes; proess known s eletroportion (Hmilton, & Sle, 1967, Devlieghere, Vermeiren, & Deevere, 2004; Wlkling-Rieiro et l., 2009). The eletril fields n e pplied for durtions vrying from nnoseonds to miroseonds, nd depending on the eletril field strength, pulse durtion nd pulse numer, permeilistion n e either reversile or irreversile (Aronsson, Borh, Stenlof, 4

6 & Ronner, 2004). The effetiveness of PEF tretment for the purpose of miroil intivtion is refletion of these prmeters (i.e. EFS nd overll tretment time). In generl, Grm-positive teri re more resistnt to tretment with PEF thn Grm negtive teri, while yests re more sensitive thn teri (Devlieghere, Vermeiren, & Deevere, 2004) The ojetive of the present study ws to ompre the effiy of MTS/PEF when pplied t two tempertures (37 C nd 55 C) to therml psteuristion. After milk hd een sujeted to proessing with MTS/PEF or high temperture short time (HTST) psteuristion, speifi miroorgnisms were monitored over 21-dy period, nd omprisons mde etween the miroiologil qulities of eh milk smple fter proessing Mterils nd Methods Milk Souring nd Preprtion Fresh rw ovine milk from ulk tnk ws otined from Lyons Reserh Frm (Newstle, Co. Dulin) nd trnsported to the lortories t UCD Dulin within 40 min. Three thes of rw whole milk were otined on three seprte osions. Milk ws stored t 4 C until required. On the sme dy s olletion from the frm, milk ws preheted to 55 C nd homogenized t totl pressure of 21 MP. A two-stge high-pressure homogenizer (Model No. Pnd 2682, Niro Sovi, Prm, Itly) ws used for this purpose, nd pressures of 18 MP nd 3MP were pplied in the primry nd seondry stges, respetively. Milk ws susequently proessed using non-therml methods or onventionl psteuristion. 2.2 Tretment of Milk with MTS/PEF Previous experiments in this lortory exmining the inhiitory effet of (i) MTS nd (ii) PEF s individul tretments showed tht optiml miroil intivtion levels were hieved 5

7 using power levels of 27.9 µm (225 kp) nd 32 kv/m for MTS nd PEF, respetively (dt not shown). Thus, these were the power settings used in the present study. Other prmeters pplied were s follows: for MTS tretment, the frequeny ws set to 20kHz, nd the totl pressure ws 225kP, while for PEF the frequeny ws set t 320kHz, nd the pulse width ws 10 µs. A peristlti pump (Msterflex L/S, Model No , Cole-Prmer Instrumentl Compny, IL, USA) ws used to pss milk through sumerged oil in heted wter th so tht the milk rehed temperture of 37 C or 55 C prior to entering the sonitors. This heting proess ws quite rpid, tking less thn one or two minutes to reh 37ºC or 55ºC, respetively. A flow rte of 160 ml/min ws used. For MTS proessing, two ultrsoni proessors were employed (Model No. UIP 1000hd, Hielsher, Germny). These sonitors were onneted in row, nd hd n opertionl frequeny of 20 khz. Two sonotrodes (Model No. BS2d40, Hielsher), eh of whih hd 40 mm frontl fe dimeter were used. Also, oosters were used to strengthen the mplitude (Model No. B2-1.8, Hielsher). The volume of eh sonition hmer ws 168ml, nd the totl residene time of milk within the hmer ws determined to e on verge 2.1 min; this durtion ws found to e dequte for miroil intivtion in preliminry studies rried out to optimise proessing onditions (dt not shown). In ddition, the hmers of eh sonitor were surrounded y jkets whih ontined flowing wter so tht the temperture within the hmer ould e mintined t 37 C or 55 C. The tempertures of the milk entering nd leving the sonitors were monitored using T-type thermoouples (Model No. SQ2020, Grnt Instruments, Cmridge, UK). Immeditely following tretment with MTS, milk ws sujeted to PEF tretment. A ontinuous lortory sle PEF unit (ELCRACK HVP 5, DIL, Germn Institute of Food Tehnologies, Qukenruk, Germny) ws used. The tretment module onsisted of three 6

8 o-liner tretment hmers with refrigerted ooling module integrted; these ooling setions were present following the first nd third hmers. Eh hmer held two oliner stinless steel eletrodes seprted y 5.0 mm gp, with the eletrode dimeter eing 3.0 mm, whih resulted in totl tretment volume of m 3. PEF tretment ws pplied using monopolr squre-wve pulses (10 µs pulse width, 320 Hz frequeny). The EFS ws 32kV/m nd the totl tretment time ws determined to e 127 µs. The produt temperture ws reorded using thermoouples (Testo 925, type-k proe, Testo AG, Lenzkirh, Germny) oth efore nd fter the tretment module, nd ws never llowed to exeed 37ºC or 55ºC depending on the tretment eing pplied. Following tretment with MTS/PEF, milk ws olleted in sterile glss ottle nd stored t 4 C until required. 2.3 Therml Psteuristion of Milk Rw, homogenised milk ws thermlly psteurised using tuulr het exhnger (Model No. FT74 UHT/HTST Proessing System, Armfield Tehnil Edution Co. Ltd, Ringwood, U.K.). Milk ws heted t 72 C for 20 s. An tthed ooling system ensured the temperture of the milk ws elow 10ºC fter tretment. Milk ws olleted in sterile glss ottle, nd stored t 4 C for miroil nlysis for the durtion of the miroiologil ssessment. 2.4 Miroiologil Anlysis The miroil ontent of ll milk smples (i.e. rw nd post-proessing with MTS/PEF or therml psteuristion) ws monitored t defined time points (dys 0, 2, 5, 7, 9, 14, 21). The totl vile ount (TVC) ws quntified y prepring deiml dilutions of eh milk smple in sterile qurter-strength Ringer s solution (Oxoid, Bsingstoke, U.K.). An liquot (100 µl) of pproprite dilutions ws then spred-plted onto plte ount gr (PCA; Shrlu, Brelon, Spin) in duplite, nd pltes were inuted t 37 C for 48 hours. The following miroorgnisms were lso quntified: 7

9 () Yests nd moulds: on potto dextrose gr (PDA; Oxoid; Bsingstoke, U.K.), inuted t 30 C for 48 h. () Stphyloous ureus: on Bird Prker gr (BPA; Merk, Drmstdt, Germny), inuted t 37 C for 48 h. () Enteroteriee: seril deiml dilutions (1 ml) of milk smples were pour-plted in violet red ile ltose dextrose gr (VRBLD; Oxoid, Bsingstoke, U.K.) with n over-lyer, nd inuted t 37 C for 24 h. (d) Lti id teri: on DeMn, Rogos nd Shrpe gr (MRS; Oxoid, Bsingstoke, U.K.), inuted t 30 C for 48 h. (e) Pseudomonds: on Pseudomons speifi gr (Oxoid, Bsingstoke, U.K.) supplemented with etrimide, fuidin nd ephloridine (CFC supplement, Oxoid, Bsingstoke, U.K.), inuted t 30 C for 48 h. (f) Totl psyhrotrophi ount: serilly-diluted milk smples in sterile qurter-strength Ringer s solution (1 ml) were pour-plted in PCA with n over-lyer, nd inuted t 4 C for period of 10 dys. The quntity of psyhrotrophi teri ws monitored on dys 0, 7, 14 nd 21 only After the pproprite inution period, olonies were ounted nd the numer of olony forming units per ml (CFU/ml) ws determined for eh speifi miroorgnism in ll of the milk smples Sttistil nlysis Sttistil nlysis ws performed using the PROC GLM funtion of SAS version 9 (SAS Institute, Cry, NC). For ll tretments, dt from three different thes of milk (i.e. n=3) were nlysed. Results were onsidered signifintly different if P< Results 8

10 Firstly, tempertures of milk efore nd fter MTS nd PEF tretments were noted during eh experiment nd the verge lulted (n=3). For milk treted t 37ºC, milk entered the MTS hmer t 37ºC (± 0ºC) nd left the MTS system t 36.5ºC (± 0.5º C). The milk then ooled to 25.7º C (± 0.5º C) efore entering the PEF system, nd the verge temperture post-pef proessing ws 32.1ºC (± 3ºC). In the se of milk treted t 55ºC, milk entered the MTS system t this temperture (± 0ºC), nd left the MTS hmer t 57ºC (± 0.4ºC). The milk susequently ooled, efore entering the PEF system t temperture of 40.5ºC (± 0.4º C). Tempertures post-pef tretments were found to e on verge 32.1ºC (± 5.1ºC). All proessed milks (i.e. 37 C or 55 C MTS/PEF-treted nd thermlly psteurised) signifintly redued the TVC of milk when ompred to rw milk (P<0.05) over the 21-dy period (Figure 1). On dys 5, 7 nd 9 the TVC of milk treted t 37 C ws not found to e signifintly different from the TVC of milk tht hd undergone onventionl psteuristion (P>0.05). However, fter 21 dys, the 37 C nd 55 C MTS/PEF tretments were found to hve signifintly higher TVC thn milk tht hd een psteurised using the HTST method (P<0.05). The TVC of rw milk ws found to e CFU/ml fter 21 dys. The verge TVC of milks treted t 37 C nd 55 C fter 21 dys were found to e nd , respetively. These vlues were signifintly (P<0.05) higher thn tht of 209 psteurised milk ( CFU/ml). Nonetheless, oth the 37 C nd 55 C MTS/PEF tretments resulted in. 4 log redution of TVC when ompred to rw milk fter 21 dys. However, therml psteuristion resulted in log redution of 6.8. Similrly, ll treted milks ontined signifintly (P<0.05) lower numer of yests nd moulds thn rw milk over the entire 21-dy period (Figure 2). On verge, rw milk ontined CFU/ml on dy 0, rising to CFU/ml on dy 21. All tretments redued the level of yests nd moulds in the milk over the 21-dy period. On 9

11 dys 2 nd 5, no signifint differene ws found etween ny of the tretments t the 5% signifine level. On dys 7 nd 9, no signifint differene ws found etween the 37 C MTS/PEF tretment nd onventionl psteuristion (P>0.05). By dy 21, thermlly psteurised milk ws found to ontin the lest numer of yests nd moulds (P<0.05). With regrd to S. ureus, rw milk generlly ontined signifintly higher level of this miroorgnism over the 21-dy period thn ll proessed milks (Figure 3). The verge initil level of S. ureus in rw milk ws determined to e CFU/ml, nd this level rehed CFU/ml y dy 21, with ll tretments signifintly reduing the level of S. ureus in milk (P<0.05). However, on dy 21 the 37 C MTS/PEF tretment ws found to hve similr level of S. ureus s rw milk. On dy 0, psteuristion redued the mount of S. ureus y 3 log yles when ompred to rw milk, while tretment with MTS/PEF t 37 C nd 55 C resulted in log redutions of 1.4 nd 2.7, respetively. On dys 2, 5, 7 nd 9, no signifint differenes were oserved etween the S. ureus ontent of ll treted milk smples (i.e. MTS/PEF or psteurised) t the 5% signifine level. On dys 14 nd 21, psteurised milk ws found to ontin signifintly lower levels of S. ureus thn milks treted with MTS/PEF t 37 C nd 55 C In the se of Enteroteriee (Figure 4), rw milk ontined greter levels of Enteroteriee thn ll other milks (P<0.05) t eh of the time points exmined. On dys 0, 2 nd 9, no signifint differene ws oserved etween the milks treted with MTS/PEF t oth tempertures (i.e. t 37 C nd 55 C). On dys 5, 7, 14 nd 21, no signifint differene ws oserved etween the Enteroteriee ontent of milks treted with MTS/PEF t 37 C nd onventionl psteuristion (P<0.05). On dy 21, the level of Enteroteriee in rw milk hd rehed CFU/ml, wheres the levels present in milks treted t 37 C nd 55 C with MTS/PEF were redued y 4.4 nd 4.1 log yles, 10

12 respetively. Therml psteuristion ppered to lmost ompletely prevent growth of Enteroteriee for the durtion of the study. Rw milk ws lso found to ontin signifintly more lti id teri (LAB) t eh time point thn ny of the other proessed milk smples (P<0.05) (Figure 5). On dys 2, 5 nd 7, no signifint differene (P>0.05) ws oserved etween ny of the tretments, ut on dys 14 nd 21, onventionlly psteurised milk ontined less lti id teri thn milk heted t 37 C nd 55 C prior to MTS/PEF tretment. By dy 21, rw milk ws found to ontin CFU/ml LAB, while tretments where milk ws heted to 37 C nd 55 C efore eing sujeted to MTS/PEF used log redutions of 2.9 nd 2, respetively, with onventionl psteuristion resulting in log redution of 5.4. With the exeption of dys 9 nd 14, rw milk ws found to ontin signifintly more pseudomonds thn ll of the proessed milk smples (P<0.05) (Figure 6). Also, on dys 0, 2, 5, 7 nd 9, milk whih hd een treted t 55 C followed y MTS/PEF ontined similr level of pseudomonds to thermlly psteurised milk (P>0.05). No signifint differene ws found etween milks treted t 37 C nd 55 C followed y MTS/PEF t ny of the time points over the 21 dys (P<0.05). By dy 21, the level of pseudomonds hd rehed CFU/ml, ut treting milk y heting t 37 C nd 55 C prior to MTS/PEF resulted in log redutions of 1.9 nd 1.8, respetively. However, therml psteuristion ws found to show sustntilly higher log redution (6.6) in the level of pseudomonds in omprison to rw milk thn ll other tretments y dy 21 (P<0.05). Finlly, the levels of psyhrotrophi teri in rw milk, MTS/PEF treted milk smples nd psteurised milk were mesured t four time intervls over the 21-dy period (Figure 7). As hd een determined for ll other miroil speies exmined throughout this study, rw milk ws found to ontin higher levels of psyhrotrophs thn milks whih hd een sujeted to MTS/PEF or onventionl psteuristion. The verge initil level of 11

13 psyhrotrophi teri from the three thes of rw milk ws estimted to e CFU/ml, nd this level rehed CFU/ml y dy 21. Plte ount results from dy 0 showed there ws minimum of 3.5 log redution in the level of psyhrotrophi teri in milk fter tretment with MTS/PEF. On dys 14 nd 21, thermlly psteurised milk ontined signifintly lower levels of psyhrotrophs thn milks treted with MTS/PEF t either 37 C or 55 C (P<0.05). 4. Disussion Rw milk ontins wide rnge of miroorgnisms inluding yests nd moulds, oth Grm positive nd Grm negtive teri, nd rnge of teril spores (Shmsi, Versteeg, Sherkt, & Wn, 2008). The present study hs shown tht omining two non-therml tehnologies (i.e. MTS nd PEF) n derese the miroil popultions of rw milk, nd onsequently improve the miroiologil qulity. A study y Noi et l. (2009) reported intivtion of Listeri innou y up to. 7 log yles following tretment with thermosonition immeditely followed y pulsed eletri fields. No other studies hve een undertken to ssess the miroiologil qulity of milk following tretment with omintion of mnothermosonition nd pulsed eletri fields. Thus, no diret omprisons n e mde etween the present study nd other pulished studies. However, detils of some studies whih pplied ultrsound nd pulsed eletri fields s seprte entities for the purpose of miroil intivtion hve een reported Milk proessing y high intensity pulsed eletri fields hs een reviewed previously (Bendiho, Bros-Cnovs, & Mrtin, 2002), nd mny reserhers hve reported how PEF n e used speifilly for miroil intivtion in milk (Odriozol-Serrno, Bendiho-Port, & Mrtin-Belloso, 2006; Cserhlmi, Sss-Kiss, Toth-Mrkus, & Lehner, 2006; Crven et l., 2008; Shmsi, Versteeg, Sherkt, & Wn, 2008; Sepulved, Gongor- Nieto, Guerrero, & Bros-Cnovs, 2009; Wlkling-Rieiro, Rodriguez-Gonzlez, 12

14 Jyrm, & Griffiths, 2011). In ddition, PEF is populr method for extending the shelf-life of fruit juies (Wlkling-Rieiro et l., 2009, 2009, 2010; Yeom, Streker, Zhng, & Min, 2000) nd fruit juie-milk sed everges (Wlkling-Rieiro et l., 2008; Smpedro et l., 2009; Slvi-Trujillo, Morles-de l Pen, Rojs-Gru, & Mrtin-Belloso, 2011). Therefore, omining PEF with other non-therml tehnologies ould represent n lterntive pproh to improving miroiologil qulity nd onsequently prolonging the shelf-life of mny everges, oth diry nd non-diry sed Under the experimentl onditions used in the present study for MTS nd PEF, the TVC vlues were redued onsiderly following proessing t oth tempertures (37 C nd 55 C), produing milk with similr TVC to tht of onventionlly psteurised milk. A study y Villmiel nd de Jong (2000) whih ssessed the totl teril ount of milk following tretment with ultrsound (20 khz) in ontinuous-flow system reported redutions of 0.2, 0.6 nd 2.9 log yles following tretment with US t residene times of 34, 56 nd 102 seonds, respetively, inside the tretment hmer. In ddition, the tempertures rehed during these tretments were 48.6 C, 62 C nd 76 C, respetively. These intivtion vlues re muh lower thn those of the present study, where only mild het ws pplied during MTS/PEF tretment (i.e. 37 C nd 55 C). In the sme study y Villmiel nd de Jong (2000), milks whih hd een proessed t 62 C in ontinuous flow ultrsound system or in onventionl heting system were ompred over five dy period. It ws determined tht the numer of teri remined onstnt t. 4 log CFU/ml until the third dy of storge, nd then went on to reh. 5 log CFU/ml fter five dys, with the inrese in totl teri ounts eing similr for oth US-treted milk nd thermlly treted milk. These vlues for intivtion re in ontrst to those oserved in the present study, where MTS/PEF proessed milk treted t 37 C nd 55 C hd TVC levels of. 1.8 nd 2.7 log CFU/ml, respetively, 13

15 seven dys fter tretment. This demonstrtes the enefits of omining MTS with PEF so tht higher levels of miroil intivtion n e hieved Another study y Choulir, Georgoginni, Knellopoulou, & Kontomins (2010) exmined TVC levels in full ft milk following tretment with US over n eight dy period. Ultrsound ws pplied t 24 khz (mplitude ws not speified) nd fter eight dys, it ws determined tht rw milk ontined 4.3 log CFU/ml, ut vlues were redued to 3.8, 3.1, 2.9 nd 2.9 log CFU/ml following 2, 4, 8 nd 16 mins of sonition, respetively. Similr studies hve lso een onduted y Bermudez-Aguirre nd o-workers where miroil levels of milk following tretment with US (Bermudez-Aguirre, Mwson, Versteeg, & Bros- Cnovs, 2009) nd PEF (Bermudez-Aguirre et l., 2011) were ssessed. In one study, these uthors exmined the totl mesophili ount of rw whole milk over 16-dy period (Bermudez-Aguirre, Mwson, Versteeg, & Bros-Cnovs, 2009). The mesophili ount ws determined to e 4.7 log CFU/ml on dy 0, nd fter tretment with US (24 khz, 108 µm, 63 C, 30 min) this ws redued to. 2 log CFU/ml nd <1 log CFU/ml on dys 0 nd 16, respetively. Thermlly treted milk ontined. 5.8 log CFU/ml fter 16 dys under refrigertion t 4 C. In ddition, these reserhers pplied ultrsound to milk t 36 µm, 72 µm, 108 µm nd 120 µm, nd no more thn 2 log yles of mesophiles were deteted over the 16-dy period. However, it should e tken into onsidertion tht milk ws heted t 63 C for 30 min while eing treted with ultrsound during these experiments. In the present study, ultrsound ws pplied for. 2.1 mins, nd milk ws heted t 37 C or 55 C. Immeditely fter proessing (i.e. on dy 0), milks heted t these tempertures (37 C, 55 C) during MTS tretment still yielded redutions of 2.6 nd 3.6 log yles, respetively, in spite of reltively mild nd moderte het tretments pplied With regrd to PEF, shelf-life study y Odriozol-Serrno, Bendiho-Port, & Mrtin-Belloso (2006) reported redution of mesophili teri of etween 1 nd 2 log 14

16 yles in rw whole milk treted t 35.5 kv/m, temperture of <40 C nd pulse width of 7 µs. A seprte study y Bermudez-Aguirre et l. (2011) exmined the effet of PEF (46.15 kv/m, 200 khz, 30 pulses 2 µs, flow rte 1 L/min) on miroil intivtion in whole ft milk. It ws determined tht mesophili nd psyhrotophi teri were redued y 0.4 nd 0.8 log yles, respetively, fter tretment with PEF t 40 C. It ws oserved during this study tht in generl, mesophili teri were more resistnt thn psyhrotrophi teri to PEF therml tretment, lthough intivtion of oth of these types of miroorgnisms ws not onsidered to e high. A shelf-life study rried out y Bermudez-Aguirre et l. (2011) showed tht mesophiles grew rpidly during storge t 4 C in milk fter tretment with PEF t 20 C, 30 C nd 40 C, with the level of mesophili teri exeeding 6 log yles fter the first week of storge. A similr growth pttern ws oserved for psyhrophili teri, regrdless of the PEF proess temperture (Bermudez-Aguirre et l., 2011). In ddition, shelf-life study rried out y Odriozol-Serrno, Bendiho-Port, & Mrtin-Belloso (2006) where whole rw milk ws sujeted to PEF (35.5 kv/m, 1000 µs, 111 Hz) in ontinuous flow system (60 ml/min) showed tht fter eight dys of storge, PEF-treted milk ws found to ontin similr levels of mesophili eroi miroorgnisms s milk whih hd een treted with therml psteuristion (P>0.05) Other reserhers hve reported US to hve no effet on the levels of yests nd moulds in milk. In n investigtion rried out y Engin nd Krgul-Yueer (2012), no signifint differene ws found etween the levels of yests nd moulds in US treted (20 khz, 5 C, 75 W, 15 min) milk nd rw milk (P>0.05). In this study the effet of US on Stphyloous sp. nd E. oli were lso ssessed, nd it ws determined tht under the speified onditions these miroorgnisms were redued y 0.8 nd 1.15 log yles, respetively. A seprte study y Hereg, Jmrk, Lels, & Thgrd (2012) exmined the intivtion of S. ureus nd E. oli in rw whole ft (4%) ow s milk immeditely following 15

17 tretment with US (20 khz, 120 µm). It ws determined tht fter tretment with US for 12 mins t 60 C, S. ureus nd E. oli were redued y 1.5 nd 3.1 log yles, respetively. However, in the present study, where sonition ws only pplied for pproximtely 2 mins, S. ureus ws found to e redued y 1.4 nd 2.7 log yles following tretment with MTS/PEF t respetive tempertures of 37 C nd 55 C, immeditely following proessing. The redution of E. oli s single miroorgnism ws not quntified in the present study, ut the levels of Enteroteriee were redued y 3.9 nd 3.63 log yles following tretment with MTS/PEF t 37 C nd 55 C, respetively. The levels of intivtion oserved in the urrent study were hieved using lower mplitude of US (27.9 µm ompred to 120 µm), nd shorter tretment time (. 2.1 mins). Tking into onsidertion the intivtion of S. ureus, it ppers tht lthough ultrsound lone used up to 1.49 log yles of intivtion (Hereg, Jmrk, Lels, & Thgrd, 2012) under sonition onditions different (i.e. higher mplitude, higher tretment temperture) to those pplied in the present study, greter intivtion of S. ureus ws hieved (. 2.7 log yles t 55 C) in the urrent study when MTS/PEF ws pplied in omintion. This suggests tht either the ddition of (i) pressure or (ii) PEF resulted in n inrese in the ntimiroil effiy of ultrsound A shelf-life study y Jurg, Slmon, Hereg, & Jmrk, (2011) using whole ft (4%) ow s milk showed tht fter five dys of refrigertion, the levels of Enteroterie were redued y 0.2, 2.7 nd 3.1 log yles when ompred to rw milk following tretment with US (20 khz, 120 µm) for 6, 9 nd 12 mins, respetively. These redutions were inresed to 2.12, 4.63 nd 3.05 log yles when US ws omined with het (60 C). The intivtion levels of Enteroteriee oserved in the present study were found to e fr greter ( minimum of 3.2 log yles) thn those reported y Jurg, Slmon, Hereg, & Jmrk, (2011), even though lower mplitude level ws used in our study (27.9 µm), nd lower tempertures lso (37 C nd 55 C). This shows the enefits of omining ultrsound 16

18 with pressure nd mild het (i.e. MTS), nd lso the inresed levels of intivtion whih n e hieved when MTS nd PEF tehnologies re omined A pulition y Villmiel nd de Jong (2000) disussed the effet of US (20 khz) on the viility of Pseudomons fluoresens in rw ow s milk following sonition in ontinuous flow system. Redutions of 0.6, 0.8 nd 3.1 log yles were oserved, ut tempertures of 43.1 C, 49 C nd 61.6 C were pplied, respetively, in order to hieve these levels of intivtion. Experiments previously rried out in our lortory using P. fluoresens showed fter sujeting this miroorgnism to tretment with either PEF or omintion of US/PEF, some P. fluoreseens ells were not killed ut merely su-lethlly injured (unpulished dt). This my explin why, in the present study, the levels of pseudomonds rehed suh high levels (. 7 logs) fter 21 dys following tretment with MTS/PEF t 37 C nd 55 C. Milk whih hd undergone therml psteuristion ontined only. 1 log of pseudomonds fter 21 dys, showing tht therml psteuristion kills this miroorgnism without su-lethlly injuring these teril ells. In ddition, study y Crven et l. (2008) showed tht pseudomonds ould e redued y >5 log yles following tretment with PEF (31 kv/m) nd mild het of up to 55 C. In the present study, lower intivtion vlues of pseudomonds were reorded for MTS/PEF under our experimentl onditions, ut it should e noted tht the temperture did not exeed 40 C during PEF tretment In the study y Choulir, Georgoginni, Knellopoulou, & Kontomins (2010), psyhrotrophi teri were lso quntified. On dy 0, rw milk whih hd not een sujeted to sonition ontined 3.3 log CFU/ml psyhrotrophi teri, ut these levels were redued to 3.2, 2, 2.2 nd 1.9 following 2, 4, 8 nd 16 mins of sonition, respetively (24 khz, mplitude not speified). After eight dys, milk whih hd een sonited for 16 mins ontined signifintly fewer psyhrotrophi teri thn ll other milk smples 17

19 (P<0.05). However, it should e noted tht these milk smples were not sujeted to sonition in ontinuous-flow system. In the urrent study, levels of psyhrotrophi teri rehed. 4 log CFU/ml in milk treted with MTS/PEF t 37 C nd 55 C fter 21 dys of storge t 4 C. This inrese in miroil levels my hve een due to the presene of some Grm-positive thermoduri teri nd spores, whih re known to hve the potentil to survive therml psteuristion nd/or PEF nd ultrsound proessing (Shmsi, Versteeg, Sherkt, & Wn, 2008). It my e the se tht greter numer of thermoduri teri re killed y therml psteuristion thn y proessing with mnothermosonition nd PEF, ut further investigtion would e required in order to determine the ext nture of the intivtion of suh miroorgnisms. Some preliminry studies rried out in this lortory hve shown tht tretment with MTS/PEF used su-lethl injury of E. oli K12 nd Ps. fluoresens (dt not shown), nd it my e the se tht other miroorgnisms re merely injured following tretment with these non-therml tehnologies, nd re not tully killed. Nonetheless, the present study shows tht intivtion of psyhrotrophi teri is hievle y MTS/PEF Conlusion The present study hs shown tht treting milk with MTS/PEF n inhiit miroil growth without the use of exessive het, suh s the tempertures employed during therml psteuristion. Although onventionl psteuristion ppers to e the most effetive method of prolonging the shelf-life of milk, the results for miroil intivtion following tretment with MTS/PEF re promising. However, further optimistion of proessing onditions would e required in order to improve the miroiologil qulity of milk treted with MTS/PEF so tht miroil ontent is on pr with those typil of thermlly psteurised milk. Also, it would e worthwhile to investigte the effiy of these tehnologies for ontrol of other 18

20 pthogens ssoited with rw milk, suh s Listeri monoytogenes, Yersini enterooliti nd Cmpyloter jejuni. 440 Aknowledgements This work ws supported y SMARTMILK, 2 yer R&D projet funded y the Seventh Frmework Progrmme of the EC under the Reserh for SMEs su-progrmme. Grnt greement No The uthors would lso like to thnk Lyons Reserh Frm (Newstle, Co. Dulin) for supplying the milk used throughout this study

21 446 Literture ited Aronsson, K., Borh, E., Stenlof, B., & Ronner, U. (2004) Growth of pulsed eletri field exposed Esherihi oli in reltion to intivtion nd environmentl ftors. Interntionl Journl of Food Miroiology, 93 (1), Bendiho, S., Bros-Cnovs, G.V., & Mrtin, O. (2002) Milk proessing y high intensity pulsed eletri fields. Trends in Food Siene nd Tehnology, 13, Bermudez-Aguirre, D., Mwson, R., Versteeg, K., & Bros-Cnovs, G.V. (2009) Composition properties, physiohemil hrteristis nd shelf-life of whole milk fter therml nd thermo-sonition tretments. Journl of Food Qulity, 32 (3), Bermudez-Aguirre, D., Fernndez, S., Esquivel, H., Dunne, P.C., & Bros-Cnovs, G. V. (2011) Milk proessed y pulsed eletri fields: Evlution of miroil qulity, physiohemil hrteristis, nd seleted nutrients t different storge onditions. Journl of Food Siene, 76 (5), S289-S Choulir, E., Georgoginni, K.G., Knellopoulou, N., & Kontomins, M.G. (2010) Effet of ultrsonition on miroiologil, hemil nd sensory properties of rw, thermized nd psteurized milk. Interntionl Diry Journl, 20 (5), Crven, H.M., Swiergon, P., Ng, S., Midgely, C., Versteeg, C., Coventry, M.J., & Wn, J. (2008) Evlution of pulsed eletri field nd miniml het tretments for intivtion of pseudomonds nd enhnement of milk shelf-life. Innovtive Food Siene & Tehnologies, 9 (2),

22 Cserhlmi, Z., Sss-Kiss, A., Toth-Mrkus, M., & Lehner, N. (2006) Study of pulsed eletri field treted itrus juies. Innovtive Food Siene & Emerging Tehnologies, 7, Devlieghere, F., Vermeiren, L., & Deevere, J. (2004) New preservtion tehnologies: Possiilities nd limittions. Interntionl Diry Journl, 14 (4), Doltowski, Z.J., Stdnik, J., & Stsik, D. (2007) Applitions of ultrsound in food tehnology. At Si. Pol., Tehnol. Aliment., 6 (3), Engin, B., & Krgul-Yueer, Y. (2012) Effets of ultrviolet light nd ultrsound on miroil qulity nd rom-tive omponents of milk. Journl of the Siene of Food nd Agriulture, 92, Hmilton W.A., & Sle, A.J.H. (1967) Effets of high eletri fields on miroorgnisms. II. Mehnism of tion of the lethl effet. Biohimi et Biophysi At, 87, Hereg, Z., Jmrk, A.R., Lels, V., & Thgrd, S.M. (2012) The effet of high intensity ultrsound tretment on the mount of Stphyloous ureus nd Esherihi oli in milk. Food Tehnology nd Biotehnology, 50 (1), Jurg, E., Slmon, B.S., Hereg, Z., & Jmrk, A.R. (2011) Applition of high intensity ultrsound tretment on Enteroterie ount in milk. Mljekrstvo, 61 (2), Mns, P., & Pgn, R. (2005) Miroil intivtion y new tehnologies of food preservtion. Journl of Applied Miroiology, 98 (6), Noi, F., Wlkling-Rieiro, M., Cronin. D.A., Morgn, D.J., & Lyng, J.G. (2009) Effet of thermosonition, pulsed eletri field nd their omintion on intivtion of Listeri innou in milk. Interntionl Diry Journl, 19,

23 Odriozol-Serrno, I., Bendiho-Port, S., & Mrtin-Belloso, O. (2006) Comprtive study on shelf life of whole milk proessed y high-intensity pulsed eletri field or het tretment. Journl of Diry Siene, 89, Pered, J., Ferrgut, V., Quevedo, J.M., Gumis, B., & Trujillo, A.J. (2009) Het dmge evlution in ultr-high pressure homogenized milk. Food Hydroolloids, 23 (7), Piysen, P., Mohre, E., & MKellr, R.C. (2003) Intivtion of miroes using ultrsound: review. Interntionl Journl of Food Miroiology, 87 (3), Slvi-Trujillo, L., Morles-de l Pen, M., Rojs-Gru, M.A., & Mrtin-Belloso, O. (2011) Miroil nd enzymti stility of fruit juie-milk sed everges treted y high intensity pulsed eletri fields or het during refrigerted storge. Food Control, 22 (10), Smpedro, F., Geveke, D.J., Fn, X., Rodrigo, D., & Zhng, Q.H. (2009) Shelf-life study of n ornge juie-milk sed everge fter PEF nd therml proessing. Journl of Food Siene, 74 (2), S107-S Sepulved, D.R., Gongor-Nieto, M.M., Guerrero, J.A., & Bros-Cnovs, G.V. (2009) Shelf life of whole milk proessed y pulsed eletri fields in omintion with PEF- generted het. LWT- Food Siene nd Tehnology, 42 (3), Shmsi, K., Versteeg, C., Sherkt, F., & Wn, J. (2008) Alkline phosphtse nd miroil intivtion y pulsed eletri field in ovine milk. Innovtive Food Siene & Emerging Tehnologies, 9, Villmiel, M., & de Jong, P. (2000) Intivtion of Pseudomons fluoresens nd Streptoous thermophilus in Tryptise Soy Broth nd totl teri in milk y 22

24 ontinuous-flow ultrsoni tretment nd onventionl heting. Journl of Food Engineering, 45 (3), Wlkling-Rieiro, M., Noi, F., Cronin, D.A., Lyng, J.G., & Morgn, D.J. (2008) Intivtion of Esherihi oli in tropil fruit smoothie y omintion of het nd pulsed eletri fields. Journl of Food Siene, 73 (8), M395-M Wlkling-Rieiro, M., Noi, F., Cronin, D.A., Lyng, J.G., & Morgn, D.J. (2009) Shelf life nd sensory evlution of ornge juie fter exposure to thermosonition nd pulsed eletri fields. Food nd Bioproduts Proessing, 87 (2), Wlkling-Rieiro, M., Noi, F., Riener, J., Cronin, D.A., Lyng, J.G., & Morgn, D.J. (2009) The impt of thermosonition nd pulsed eletri fields on Stphyloous ureus intivtion nd seleted qulity prmeters in ornge juie. Food nd Bioproess Tehnology, 2 (4), Wlkling-Rieiro, M., Noi, F., Cronin, D.A., Lyng, J.G., & Morgn, D.J. (2010) Shelf life nd sensory ttriutes of fruit smoothie-type everge proessed with moderte het nd pulsed eletri fields. LWT- Food Siene nd Tehnology, 43 (7), Wlkling-Rieiro, M., Rodriguez-Gonzlez, O., Jyrm, S., & Griffiths, M.W. (2011) Miroil intivtion nd shelf life omprison of old hurdle proessing with pulsed eletri fields nd mirofiltrtion, nd onventionl therml psteuristion in skim milk. Interntionl Journl of Food Miroiology, 144 (3), Yeom, H.W., Streker, C.B., Zhng, Q.H., & Min, D.B. (2000) Effets of pulsed eletri fields on the qulity of ornge juie nd omprison with het psteuriztion. Journl of Agriulturl nd Food Chemistry, 48 (10),

25 535 List of Figures Figure 1: TVC levels of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 2: Yest nd mould levels of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 3: S. ureus ontent of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 4: Enteroteriee ontent of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 5: Lti id teri levels of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 6: Pseudomonds ontent of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3). Figure 7: Psyhrotrophi teri ontent of vrious milk smples over 21-dy period; Rw milk (white r), MTS/PEF t 37 C (grey r), MTS/PEF t 55 C (striped r) nd thermlly psteurised (lk r). Dt= men ±S.D. (n=3)

26 558 Figure 1: 10 Log CFU/ml ,,,, Dy

27 562 Figure 2: 10 Log CFU/ml ,, Dy

28 565 Figure 3: Log CFU/ml ,, Dy

29 568 Figure 4: 10 9 Log CFU/ml ,, Dy

30 571 Figure 5: 10 Log CFU/ml ,, Dy

31 574 Figure 6: 10 Log CFU/ml ,,,,,,,, Dy

32 577 Figure 7: 10 Log CFU/ml ,, Dy