Iranian Journal of Fisheries Sciences 14(2)

Irnin Journl of Fisheries Sciences 14(2) 369-381 2015 Anticteril ctivities of nisin encpsulted in zein nd modified tmosphere pckging on rinow trout (Oncorhynchus mykiss) fillet during chilled storge 4 C Shmloofr M. 1 ; Hoseini E. 2 ; Kmli A. 1 ; Motlei Moghnjoghi A.A. 3 ; Poorgholm R. 4 Received: Decemer 2011 Accepted: Jnury 2012 Astrct Nisin is widely used nturlly occurring ntimicroil effective ginst mny pthogenic nd spoilge microorgnisms. It hs een proposed tht reduced efficcy of nisin in foods cn e improved y technologies such s encpsultion to protect it from interferences y food mtrix components. This study ws crried out to evlute the microiologicl qulity of fresh trout slices treted with N1 (nisin 0.15 g/kg) nd N2 (nisin 0.25 g/kg), NE1(encpsulted nisin 0.15 g/kg), NE2 (encpsulted nisin 0.25 g/kg ) nd were then pckged under Modified Atmosphere Pckging (MAP) (45% CO2, 50% N2,5% O2) nd stored t 4±1 C for 20 dys. The results reveled tht nisin in oth forms of free nd encpsulted ws efficient ginst the prolifertion of vrious ctegories of spoilge microorgnisms; including eroic nd psychrotrophic popultions nd lctic cid cteri. The shelf life of the treted products ws extended y 4 7 dys more thn tht of the control. As consequence, nisin, in prticulr encpsulted nisin, might e considered s n effective tool in preventing the qulity degrdtion of the fillet, resulting in n extension of their shelf life. Keywords: Encpsulted nisin, Anticteril ctivities, Modified Atmosphere Pckging (MAP), Rinow trout (Oncorhynchus mykiss), Chilled storge 1-Deprtment of Fisheries, Science nd Reserch Brnch, Islmic Azd University, Tehrn, Irn 2-Deprtment of Food Science nd Technology, Science nd Reserch Brnch, Islmic Azd University, Tehrn, Irn 3- Agriculturl Reserch, Eduction nd Extension Orgniztion, Irnin Fisheries Science Reserch Institute, P. O. BOX: 14155-6116 Tehrn, Irn 4- Agriculturl Reserch, Eduction nd Extension Orgniztion, Irnin Fisheries Science Reserch Institute, Cspin Se Ecology Reserch Center, Sri, Irn *Corresponding uthor s emil: Ehoseini@sriu.c.ir

370 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... Introduction Fish is n excellent protein source with high nutritive vlue due to its fvorle essentil mino cid composition nd is one of the most highly perishle food products (Jnnt Alipour et l., 2010). During hndling nd storge period, qulity deteriortion of fresh fish rpidly occurs which limits the shelf life of the product (Kshiri et l., 2011). Becuse of consumer's demnd for fresh refrigerted foods with extended shelf life, considerle reserch hs een directed towrd using vrious preservtion strtegies to prolong the shelf life, while ensuring the sfety of fresh foods including fishery products (Sllm, 2007). The ntimicroil dditives cn suppress the growth of cteri during storge with minor effects on the qulity of met products (Zhu et l., 2005). Nisin is the most commercilly importnt cteriocin tht is produced y Lctococcus lctis, nd is used extensively s sfe food preservtive (Zhu et l., 2005). It inhiits the growth nd development of mny grm positive cteri such s Listeri monocytogenes (Nyknen et l., 2000; Thoms et l., 2005). However, numerous studies hve reported much reduced ntimicroil efficiency of nisin when pplied to foods thn in growth medium. A reduction of nisin ctivity ws reported ecuse of nonspecific inding of nisin with lipids nd proteins (Delves- Broughton, 1990; Bhtti et l., 2004). Rose et l. (2008) found tht the compromised ntimicroil ctivity of nisin in fresh met ws cused y the complextion with glutthione. Incorportion of nisin within cpsules of edile polymers my reduce the interction of nisin with food components nd minimize its dysfunction in foods (Teerkrn et l., 2002; Xio nd Zhong, 2011). Slmoso et l. (2004) demonstrted tht sustined relese of nisin from poly- (L-lctide) nnocpsules inhiited the growth of Lctocillus delrueckeii over 45 dys, in comprison to c. 4 dys for unencpsulted nisin. Much work is needed to utilize GRAS (Generlly Recognized As Sfe), sustinle, nd inexpensive ingredients s delivery systems of ntimicroils nd low-cost nd sclle processes. Spry drying is quick, simple, low-cost, nd one-step method to otin powdered product nd is populr choice in the food industry to encpsulte ioctive compounds in food iopolymers (Xio nd Zhong, 2011). Zein, which is clss of lcohol-solule proteins (prolmins) extrcted from mize kernel (Zhong nd Jin, 2009), ws used for sprydried encpsulting of nisin in our study. MAP, is protecting technique used to extent the shelf-life of fish nd fish products (Özogul et l., 2006) MAP, elimintes the oxygen from inside the pckge nd fills it with different concentrtions of CO2 nd N2 (Kılınç nd Çklı., 2004). There re lot of reserch relted to the extension of the shelf life of fish with MAP, including chu mckrel (Erkn et l., 2007), rinow trout (Çklı et l., 2006; Oguzhhn nd Angis, 2012), ss (Torrieri et l., 2006), herring (Lyhs et l., 2007). The im of this reserch is to determine the comined effects of encpsulted nisin nd MAP on the shelf life of refrigerted (4ºC) rinow trout fillets y evluting certin microiologicl prmeters. Mterils nd methods

Irnin Journl of Fisheries Sciences 14(2) 2015 371 Preprtion of encpsulted nisin The 2.5% nisin ws purchsed from (Serv, USA). The product specifictions indicte nisin content of 2.5% nd 1,000 IU/mg solids, 75% sodium chloride, nd 22.5% dentured milk solids (Xio, 2010). Zein ws purchsed from (Fluk, BCBG 3298, Germny). Other chemicls were otined from (Merck, Germny). Encpsultion y spry drying The 2.5% nisin ws suspended t concentrtion of 6 mg solids per ml of 50%v/v queous ethnol. After mixing for 6 hours using stirring plte, the suspension ws centrifuged t 1,520 g for 5 min (Refrigertor Centrifuge Kokusn. h- 103nr, Jpn). The trnsferred superntnt (extrct) ws constituted to 70%v/v ethnol to dissolve zein t concentrtion of 2%w/v. Spry dryer (L-plnt UK Ltd YO14 OPH, Englnd ) ws used to dry the solution t feed rte of 5.26 ml/min nd n spirtor setting of 100% (Xio nd Zhong, 2011 ). The inlet nd outlet temperture in this study ws 105 nd 68 C, respectively. Evlution of encpsultion properties Spry-dried smples were evluted for these prmeters (Xio nd Zhong, 2011): totlnisin units in the feed (1) Encpsultion efficiency % = 100 totlnisin units in collected product non solvent mss in the feed (2) Mssyield % = 100 mss of collected product In vitro relese kinetics 1.5 ml micro-centrifuge tues were used for relese studies y suspending 4 mg spry-dried prticles in 1 ml of 20 mm sodium phosphte uffer tht ws predjusted to ph 6.0. Micro-centrifuge tues were ttched to n end-to-end shker (Fter Rizprdz, Irn) nd were continuously rotted t room temperture. Then, smples were centrifuged t 14,500 g for 5 min (Refrigertor Centrifuge Kokusn. h-103nr, Tokyo, Jpn), nd 700 μl superntnt ws smpled to determine nisin ctivity using the method elow. 700 μl of the corresponding fresh phosphte uffer ws supplemented to the remining smple tht ws then re-suspended for continued relese studies. The cumultive relese of nisin t certin time point ws clculted using the following (Xio nd Zhong, 2011): (3) Rti(%) i-1 n-1 n 10 i 7 100% u0 Where Rti (%) is the cumultively relesed nisin t time ti, the ith time of smpling; i is the nisin concentrtion (IU/mL) in the superntnt t the smpling time ti; nd Uo is the totl nisin ctivity units in 4 mg powder (corresponding to 100% relese). Determintion of nisin ctivity Nisin ctivity of smples ws determined y the stndrd gr diffusion ssy (Wolf nd Gions, 1996) using Micrococcus luteus ATCC 10240 s n indictor

372 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... microorgnism. To constitute nisin stndrd solutions, stock solution ws prepred y dissolving 0.1 g of the 2.5% nisin preprtion in 10 ml of 20 mm HCl, i.e., 10,000 IU/mL. 20 mm HCl or different volumes of ethnol nd sterile wter were used to dilute the stock solution to nisin concentrtion of 50-1500 IU/mL. Nisin solutions were loded into wells of gr gels nd incuted t 35 ºC for 24 h. Inhiition zone dimeters in gr gels corresponding to stndrd solutions were mesured nd used to generte semi-log plot, nd liner regression from the plot resulted in stndrd curve tking the form of: (4) D = log10 [Nisin] + Where D is the dimeter of the inhiition zone fter seline sutrction; [Nisin] is the concentrtion of nisin in IU/ml; nd re the slope nd intercept from the liner regression, respectively. Nisin smples prepred from encpsultion products were incuted together with stndrd solutions t 35ºC for 24h. Two smple replictes were used, ech loded in 4 well replictes in gr gels. The verge of 8 inhiition zone dimeters from ech smple ws used to estimte nisin ctivity using n pproprite stndrd curve (Xio, 2010). Preprtion nd tretment of fish smples Rinow trout (O. mykiss) (250±25g) were otined from locl mrket (Sri, North of Irn) nd trnsported to the lortory in oxes contining ice. Upon rrivl, the fish were eheded, gutted nd wshed with tp wter severl times to remove the lood nd slime. Then the fish were filleted mnully. Solutions corresponding to ech tretment of nisin for the experimentl design were prepred, nd 1 ml of ech ws spryed uniformly on the surfce of the fillets. Tretments included: C (control smples), N1 (nisin 0.15 g/kg) nd N2 (nisin 0.25 g/kg), NE1 (encpsulted nisin 0.15 g/kg), nd NE2 (encpsulted nisin 0.25 g/kg). Ech group included 18 fillets. All filleted smples, including the control, were pckged in 15x25 cm low density polyethylene/polymide/low density polyethylene (LDPE/PA/LDPE) rrier pouches. The MAP gs mixture used ws 45%/50%/5% (CO2/N2/O2). Pouches were het seled using vcuum seler (Multivc, Germny) nd kept under refrigertion (4±1 C) nd smples were sujected to microiologicl (eroic plte count, psychrotrophic cteri, lctic cid cteri,) nlyzes on certin dys (0, 4, 8, 12, 16 nd 20th dys) of storge. Microiologicl Anlysis A smple (5 g) ws tken from ech trout fillet, trnsferred septiclly into stomcher g contining 45 ml of 0.1% peptone wter, nd the mixture ws homogenized for 60 s using Stomcher (L Stomcher Blender 400-BA 7021 Sewrd medicl, Englnd) t room temperture. For microil enumertion, 0.1 ml smples of seril dilutions (1:10, diluent, 0.1% peptone wter) of fish homogentes were spred on petri dish of vrious gr mterils. Aeroic plte count Aeroic plte counts (APC) were determined y inoculting 0.1 ml of the smple homogente, t selected dilutions, onto duplicte sterile pltes of pre-poured nd dried Stndrd Method Agr (Nissui

Irnin Journl of Fisheries Sciences 14(2) 2015 373 Phrmceuticl Co,.Ltd., Tokyo, Jpn) using the surfce spred technique, then the pltes were incuted for 48 h t 35 C sed on the method descried y Americn Pulic Helth Assocition, APHA (1992). Psychrotrophic count Psychrotrophic counts (PTCs) were determined in similr method to tht for APC except tht pltes were incuted t 7 C for 10 dys (Sllm, 2006). Lctic cid cteril count To determine the lctic cid cteri (LAB), diluted smples were plted on (demn, Rogos, nd Shrpe( MRS gr (Merck, Drmstdt, Germny) nd incuted t 30 C for 2 3 dys in neroic jrs with disposle Anerocult C gs (Merck, Drmstdt, Germny) in order to generte n neroic medium. (Chytiri et l., 2004). Micriiologicl exmintions were done s triplictes nd were expressed s log10 cfu/g. Sttisticl Anlysis All the dt were presented s mens±stndrd error. The experimentl design ws fctoril 5 6 3 (4 tretments nd control, six storge times nd three replictes). All otined dt from this study were sujected to nlysis of vrince (ANOVA), followed y Duncn s multiple rnge test to determine significnt differences mong mens t (p<0.05) level, using SPSS. Results Encpsultion properties Prmeters defined to evlute encpsultion performnce of smples produced y spry-dryer re summrized in Tle 1. The encpsultion efficiency of nisin encpsulted in zein in this study, ws estimted t out 49 %. Another prmeter to evlute encpsultion performnce is mss yield tht is defined in Eqution 2. The mss yield of spry- dried zein cpsules co-encpsulted with nisin in this study ws 62 %. The relese kinetics of nisin from zein cpsules produced y spry-drying is shown in Fig 1. The vlues for this fctor t time intervls of 0, 24, 48, 72 nd 200 hours ws 42, 49, 61, 72 nd 76 %, respectively. Tle 1: Encpsultion performnce of smples produced y spry-dryer. Inlet temperture Mss yield (%) Encpsultion efficiency (%) (C ) 105 62±0.93 49.89±0.76 Aeroic plte count Tle 2 presents the chnges of APCs in ll tretments through the storge time. The initil APC (log10 CFU/g) in sliced trout rnged from 3.77 in (N1) to 3.93 in control tretment nd there ws no significnt difference (p<0.05) etween ll of the treted smples nd the control. By dy 12 of storge, however, APCs in sliced trout for ll of the different tretments were still elow 6 log10 CFU/g, while tht in controls ttined count of 6.83, which is in close proximity to the mximl recommended limit of 7 log10 CFU/g for APC in rw fish y Interntionl Commission on Microiologicl Specifiction for Foods, ICMSF (1986), indicting microiologicl shelf life of less thn 16 dys (13-14) dys for the non-treted

374 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... control smples tht were pcked under MAP. Cumultive relese% 80 70 60 50 40 30 20 10 0 0 24 48 72 200 Time (hour) Figure 1: Relese kinetics of nisin from zein cpsules produced y spry-drying. At the end of dy 20, eroic plte counts were higher thn the cceptility levels in ll control or treted fillets. Results lso showed tht with increse of the concentrtions of nisin, APCs were lower thn in the control ut greter inhiition of totl cteri ws oserved when encpsulted nisin tretment ws used. The reductions of APC for N1, N2, NE1 nd NE2 compred to the control were 0.43, 0.59, 0.95 nd 1.48 log CFU/g on dy 20 respectively. Significnt differences (p<0.05) were detected etween ll of the treted smples nd the control, nd lthough there ws no significnt difference etween N1 nd N2, ut significnt differences were detected mong the two tretments nd NE1 nd NE2. According to the results of the APCs for ll the tretments nd the mximl recommended limit of 7 log10 CFU/g for APC in rw fish the shelf life of the control, N1, N2, NE1 nd NE2 ws pproximtely 12, 16, 17, 19 nd 20 dys, respectively. Tle 2: Effects of N1(nisin 0.15 g/kg) nd N2 (nisin 0.25 g/kg), NE1(encpsulted nisin 0.15 g/kg ), nd NE2 (encpsulted nisin 0.25 g/kg ) on eroic plte counts (APC) of filleted rinow trout during storge t 4 C. Vlues represent mens±se of three replictes. Storge time (dy) Tretment 0 4 8 12 16 20 Control 3.93±.16 5.08±.042 6.10±.063 6.83±.113 7.80±.091 8.60±.113 NE1 NE2 N1 N2 3.90±.08 ±.077 3.88±.04 ±.021 3.83±.12 ±.16 c c 4.21 4.98±.084 6.15±.657 6.40±.028 7.65±.035 d d 4.13 4.88±.035 5.76±.106 6.17±.084 7.22±.035 3.93 5.04±.091 6.00±.12 6.76±.35 8.17 ±.049 3.77±.91 4.24±.176 4.90±.077 5.84±.162 6.64±.014 8.01±.134 Different superscripts within column indicte significnt differences (p< 0.05) Psychrotrophic cteri Chnges of PTCs in ll tretments through the storge time re shown in Tle 3. In this study, the initil PTCs (dy 0) of filleted trout rnged from 3.8 log10 CFU/g, in NE1 nd control to 3.66 log10 CFU/g in N2. Additionlly, the growth pttern of PTC showed sme ehvior s tht of APC with the control eing the highest on dy 20 (8.55 log10 CFU/g), followed y smples treted with N1 (7.79 log10 CFU/g), N2 (7.65 log10 CFU/g) nd NE1 (7.61 log10 CFU/g), while lower count (7.28 log10 CFU/g) ws detected in smples treted with NE2. The results did not revel ny significnt differences (p>0.05) in the

Irnin Journl of Fisheries Sciences 14(2) 2015 375 initil PTC mong different tretments or etween the treted nd control smples. However, y the end of storge (dy 20), significnt differences (p<0.05) were detected etween ll the treted trout smples nd the control. Although there ws significnt difference etween NE2 nd other tretments, no significnt differences were detected mong the PTC in N1, N2 nd NE1 treted smples. According to the results of the PTCs for ll the tretments nd the mximl recommended limit of 7 log10 CFU/g for PTC in rw fish the shelf life in the control, N1, N2, NE1 nd NE2 ws pproximtely 12, 16, 16, 18 nd 19 dys, respectively. Tle 3: Effects of N1 (nisin 0.15 g/kg) nd N2 (nisin 0.25 g/kg), NE1 (encpsulted nisin 0.15 g/kg ), NE2 (encpsulted nisin 0.25 g/kg ) on psychrotrophic counts (PTC) of filleted rinow trout during storge t 4 C. Vlues represent mens±se of three replictes. Storge time (dy) Tretment 0 4 8 12 16 20 Control 3.80±.063 4.84±.014 5.88±.056 6.885±.035 7.83±.042 8.655±.091 NE1 3.80±.0141 4.69±.014 c 5.325±.077 d 6.225±.120 c 6.775±.021 d 7.615±.063 NE2 3.68±.091 4.63±.056 c 5.25±.176 d 6.025±.049 d 6.525±.169 e 7.285±.063 c N1 3.66±.091 4.78±.084 5.62±.049 6.465±.063 7.085±.035 7.790±.098 N2 3.74±.049 4.75±036 c 5.49±.028 c 6.365±.035 6.950±.042 c 7.655±.049 Different superscripts within column indicte significnt difference (p< 0.05) Lctic cid cteril count Tle 4 presents the chnges of LABs in ll tretments through the storge time. The initil count of this cterium ws etween 2.26 nd 2.47 log cfu/g. Until the 20th dy of storge, lctic cid cteri counts did not rech 7 log cfu/g which is the mximl recommended limit for LABs count in fish fillets (ICMSF, 1986 ; Sllm, 2007) except in controls (7.26 log cfu/g). The LAB counts t the end of the storge time in the smples treted with N1, N2, NE1 nd NE2 ws (6.53, 6.27, 5.89 nd 5.93 log10 CFU/g), respectively. The results did not revel ny significnt (p>0.05) differences in the initil LAB count mong the different tretments or etween the treted nd control smples; however y the end of storge (dy 20), significnt (p<0.05) differences were detected etween ll of the treted trout smples nd the control. Although there ws significnt difference etween N1, N2 nd NE1 nd NE2, no significnt differences were detected mong the LAB counts in NE1 nd NE2 treted smples.

376 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... Tle 4: Effects of N1 (nisin 0.15 g/kg) nd N2 (nisin 0.25 g/kg), NE1(encpsulted nisin 0.15 g/kg ), NE2 (encpsulted nisin 0.25 g/kg ) on lctic cid cteri counts (LAB) of filleted rinow trout during storge t 4 C. Vlues represent mens±se of three replictes. Storge time (dy) Tretments 0 4 8 12 16 20 Control 2.41±.014 3.725±.049 4.71±.028 5.14±.0141 6.41±.233 7.26±0.162 NE1 2.46±.063 3.075±.049 4.03±.077 c 4.58±.056 c 5.52±.091 c 5.89±.021 d NE2 2.39±.028 2.8±.056 c 3.81±.063 d 4.42±.035 c 5.17±.084 d 5.93±.014 d N1 2.33±.169 3.16±.084 4.27±.049 4.81±.155 5.94±.049 6.535±.212 N2 2.65±.063 3.10±0141 4.09±.028 c 4.76±.049 5.81±.028 c 6.27±.049 c Different superscripts within column indicte significnt difference (p< 0.05) Discussion The results of this study in the field of encpsultion efficiency ws in ccordnce with tht of Xio (2010) who studied encpsultion of nisin in zein microcpsules t four inlet tempertures of spry- dryer etween 75 nd 120 C. The results reveled tht spry drying is n efficient nd simple method to encpsulte nisin. At 95 C nd ove, no pprent loss of nisin ctivity ws noticed fter spry drying nd the cpsules produced t n inlet temperture of 105 C showed the most sustined relese of nisin t ph 6.0. As we wnted to use encpsulted nisin s n ntimicroil gent to extend the shelf life of rinow trout fillets nd the ph of fish fillets ws close to 6, we selected n inlet tempertue of 105 C for the spry drier. The two importnt fctors impcting relese profiles, re cpsule structure nd moleculr interctions etween the crrier polymer nd the encpsulted compound (Zhong nd Jin, 2009). The isoelectric point (pi) of zein is 6.8 (39) nd tht of nisin is 8.8 (Miserendino et l., 2008). In this study, when ph ws 6.0, less thn 100% of nisin relese ws oserved for ll cpsules, possily due to the significnce of hydrophoic interctions ecuse oth nisin nd zein re more hydrophoic t ph 6.0. It hs een suggested tht nisin disrupts cell memrne ctivity vi pore formtion nd my hve dditionl effects on electron trnsfer chin components (De Vuyst nd Vndmme, 1995, Shirzinejd et l., 2010). Moreover, the outcome of nisin ctivity within food system depends on numerous fctors; Nture of the food, other preservtive hurdles such s het tretments, low wter ctivity, modified tmosphere, low temperture, nd ph enhnced ctivity (Szo nd Chill, 1998; Shirzinejd et l., 2010). The increse in net chrge of cteriocins t low ph might fcilitte trnsloction of cteriocin molecules through the cell wll. The soluility of cteriocins my lso increse t lower ph, fcilitting diffusion of cteriocin molecules. It should e noted tht the ntimicroil properties of CO2 under MAP re ttriuted to the reduction of ph elow the level t

Irnin Journl of Fisheries Sciences 14(2) 2015 377 which the growth of mny cteri is inhiited (Smelis et l., 2005). Undissocited wek cids possess the ility to cross memrnes of microorgnisms nd ecome dissocited inside the cell nd cidify the cell interior. A modertely high CO2 concentrtion in the tmosphere of pckges intercts positively with nisin, therey enhncing the effectiveness of this dditive. However, severl interctions etween CO2 nd nother dditive hve een demonstrted in vitro (CO2 nisin) (Nillson, 2000) nd in vivo (CO2 nisin), (Co, 2005). A possile hypothesis to explin the interctions etween CO2 nd nisin my relte to grdul chnge in the distriution of Grm-positive nd Grm-negtive orgnisms in the popultion. Considering tht nisin is more ctive ginst Grmpositive cteri (Co et l., 2005) nd tht microil flor chnge from Grmnegtive (initilly predominnt) to Grm positive when refrigerted fish is pckged under high CO2 concentrtions, n increse in their effect with incresing CO2 is expected (Co et l., 2005). The initil qulity of fish used in this study ws good, s indicted y low initil numer of cteri (<4 log10 CFU/g) efore the fish slices were sujected to the different tretments. Chytiri et l. ( 2004), report n initil APC of whole ungutted nd filleted rinow trout were c. 2.5 nd 3.8 log cfu/cm2, respectively (dy 0). In their study APC reched c. 7.0 log cfu/cm 2 fter 18 dys of storge for whole ungutted trout nd fter 10 dys for filleted smples. The results indicted tht nisin lone ws less effective on APCs growth when compred with encpsulted nisin with zein cpsules. Nisin lone or in comintion with lctic cid ws effective in reducing the APC when compred to the control (Gogus et l., 2006). In cold smoked rinow trout, nisin ws effective in reducing the count of totl plte count (Nyknen et l., 2000). This greter inhiition of NE1 nd NE2, compred to the N nd N2, my e due to the reduction of free nisin ctivity, nd it ws due to the nonspecific inding of nisin with lipids nd proteins (Bhtti et l., 2004). The Grm-negtive psychrotrophic cteri re the mjor group of microorgnisms responsile for spoilge of eroiclly stored fresh fish t chilled tempertures (Mohn et l., 2010). The results of this study were in ccordnce with tht of Hozor et l. (2006), who reveled similr growth ptterns for psychrotrophic popultion in se slmon during refrigerted storge. The results indicted tht nisin lone ws less effective on PTCs growth when compred with encpsulted nisin with zein cpsules. Rose et l. (2008) found tht the compromised ntimicroil ctivity of nisin in fresh met ws cused y the complextion with glutthione. Incorportion of nisin within zein cpsules in this study my hve cused reduction in the interction of nisin with food components, nd thus encpsulted nisin produced etter ntimicroil results. Lctic cid cteri cn reduce the qulity of fish nd fish products nd re usully ssocited with the spoilge of fish (Mohn et l, 2010). The low LAB count in this study ws expected since lctic cid cteri tend to grow slowly t refrigertion tempertures nd re under eroic conditions generlly out-competed y

378 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... pseudomons (Shirzinejd et l., 2010). In contrst, the contriution of LAB s the mjor spoiling microorgnism hd een reported in fresh vcuum-pcked Atlntic slmon portions stored t 4 C (Rsmussen et l., 2002) Grdul increse in the counts of lctocillus ws oserved with increse in the storge time. This result ws in ccordnce with tht of Fghni et l. (2011), who reveled similr growth ptterns for LABs in grss crp Ctenophryngodon idell treted with nisin nd sodium cette during refrigerted storge. The results showed tht encpsulted nisin tretments were more effective in reducing the LAB counts, compred with free nisin tretments. In conclusion, nisin in the form of free nd encpsulted ws effective in reducing microil counts nd retrding the oxidtion process in rinow trout fillets under refrigertion storge nd lso the results of this study hve clerly shown tht interctions etween CO2 nd nisin re n dvntge in the ppliction of MAP in food preservtion. Acknowledgments This study ws crried out in the Cspin Se Ecology Reserch Center, Sri. The uthors grtefully cknowledge the efforts of ll collegues in the Biotechnology Dept. nd mny thnks to ll people tht helped in performing this reserch. References Americn Pulic Helth Assocition, 1992. In C. Vnderznt & D.F. Splittsloesser (Eds.), Compendium of methods for the microiologicl exmintion of foods (3 rd ed.). Wshington, DC. Bhtti, M., Veermchneni, A. nd Shelef, L.A., 2004. Fctors ffecting the nti-listeril effects of nisin in milk. Interntionl Journl of Food Microiology, 97, 215-219. Co, M.L., Herrer, J.J.R., Smpedro, G. nd Pstoriz, L., 2005. Appliction of nisin, CO2 nd permeilizing gent in the preservtion of refrigerted lue whiting (Micromesistius poutssou). Journl of the Science of Food nd Agriculture, 85, 1733 1740. Chytiri, S., Choulir, I., Svvidis, I.N. nd Kontomins, M.G., 2004. Microiologicl, chemicl nd sensory ssessment of iced whole nd filleted qucultured rinow trout. Journl of Food Microiology, 21, 157 165. De Vuyst, L. nd Vndmme, E.J., 1995. Nisin, n ntiiotic Produced y Lctococcus lctis susp. Lctis: properties, iosynthesis, fermenttion nd pplictions. In: Bcteriocins of lctic cid cteri: Microiology, genetics nd pplictions (Edited y L. De Vuyst & E.J. Vndmme). Pp. 151 221. London: Blckie Acdemic nd Professionl. Delves-Broughton, J., 1990. Nisin nd its uses s food preservtive. Food Technology, 44, 100-117. Erkn, N., Özden, Ö. nd Inugur, M., 2007. The effects of modified tmosphere nd vcuum pckging on qulity of chu mckerel. Interntionl Journl of Food Science nd Technology, 42, 1297-1304.

Irnin Journl of Fisheries Sciences 14(2) 2015 379 Fghni Lngroudi, H., Soltni, M., Kmli, K., Ghomi, M.R., Hoseini, S.E., Benjkul, S. nd Heshmtipour, Z., 2011. Effect of Listeri monocytogenes inocultion, sodium cette nd nisin on microiologicl nd chemicl qulity of grss crp Ctenophryngodon idell during refrigertion storge. Africn Journl of Biotechnology, 10(42), 8484-8490. Gogus, U., Bozoglu, F. nd Yurdugul, S., 2006. Comprtive effects of lctic cid, nd nisin, coting comined nd lone pplictions on some postmortem qulity criteri of refrigerted Srdin pilchrdus. Journl of Food Qulity, 29, 658-671. Hozor, M.C., Siz, A.I., Yennes, M.I. nd Fritz, R., 2006. Microiologicl chnges nd its correltion with qulity indices during eroic iced storge of se slmon (Pseudopercis semifscit). LWT Food Science nd Technology, 39, 99 104. Interntionl Commission on Microiologicl Specifiction for Foods, 1986. Microorgnisms in foods. 2. Smpling for microiologicl nlysis: principles nd specific pplictions (2 nd ed). Bufflo, NY: University of Toronto Press. Jnnt Alipour, H., Shnpoor, B., Shni, A. nd Sdeghi Mhoonk, A., 2010. Effects of cooking methods on physico-chemicl nd nutritionl properties of Persin sturgeon )Acipenser persicus( fillet. Interntionl Aqutic Reserch, 2, 15-23. Kshiri, H., Hghprst, S. nd Shnpour, B., 2011. Effects of sodium slt solutions (sodium cette, lctte nd citrte) on physicochemicl nd sensory chrcteristics of Persin Sturgeon (Acipenser persicus) fillets under refrigerted storge. Journl of Agriculturl Technology, 13, 89-98. Kılınç, B. nd Çklı. S., 2004. Su ürünlerinin modifiye tmosferde pketlenmesi. Journl of Fisheries nd Aqutic Sciences, 21(3-4), 349-353. Lyhs, U., Lhtinen, J. nd Schelvis- Smit, R., 2007. Microiologicl qulity of mtjes herring stored in ir nd under modified tmosphere t 4 nd 10 C. Food Microiology, 24, 508-516. Miserendino, T., Demirci, A. nd Pongthrngkul, T., 2008. Nisin production y immoilized microil cell culture during tch nd fed tch fermenttions with vrious ph profiles. Agriculturl Engineering Interntionl, 10, 1-17. Mohn, C.O., Rvishnkr, C.N., Gopl, T.K.S., Llith, K.V. nd Kumr, K.A., 2010. Effect of reduced oxygen tmosphere nd sodium cette tretment on the microil qulity chnges of seer fish (Scomeromorus commerson) steks stored in ice. Food Microioogyl, In press. Nyknen, A., Weckmn, K. nd Lpvetlinen, A., 2000. Synergistic inhiition of listeri monocytogenes on cold-smoked rinow trout y nisin nd sodium lctte. Interntionl Journl of Food Microiology, 61, 63-72.

380 Shmloofr et l., Anticteril ctivities of Nisin encpsulted in... Oguzhhn, P. nd Angis, S., 2012. Effect of slting nd pckging tretments on fresh rinow trout (Oncorhynchus mykiss) fillets during storge t refrigertor tempertures. J.Vet Fk Derg, 28, 54-63. Özogul, Y., Özogul, F. nd Küley, E., 2006. Modifiye edilmis tmosfer pketlemenin lık ve lık ürünlerine etkisi. Journl of Fisheries nd Aqutic Sciences, 23(1-2), 193-200. Rsmussen, S.K.J., Ross, T., Olley, J. nd Mcmeekin, T., 2002. A process risk model for the shelf life of Atlntic Slmon fillets. Interntionl Journl of Food Microiology, 73, 47 60. Rose, N.L., Sporns, P., Stiles, M.E. nd Mc Mullen, L.M., 2008. Inctivtion of nisin y glutthione in fresh met. Journl of Food Science, 64, 759-762. Sllm, K.I., 2006. Antimicroil nd ntioxidnt effects of sodium cette, sodium lctte, nd sodium citrte in refrigerted sliced slmon. Journl of Food Control, 18(5), 566-575. Sllm, K.I., 2007. Chemicl, sensory nd shelf life evlution of sliced slmon treted with slts of orgnic cids. Food Chemistry, 101, 592-600. Slmso, S., Elvssore, N., Bertucco, A., Lnte, A. nd Cliceti, P., 2004. Nisin-loded poly-l-lctide nnoprticles produced y CO2 nti-solvent precipittion for sustined ntimicroil ctivity. Interntionl Journl of Phrmceutics, 287(1-2), 163-173. Smelis, J., Bedie, G.K., Sofos, J.N., Belk, K.E., Scng, J.A. nd Smith, G.C., 2005. Comintions of nisin with orgnic cids or slts to control Listeri monocytogenes on sliced pork ologn stored t 4 C in vcuum pckges. LWT Food Science Technology, 38, 21 28. Shirzinejd, A., Noryti, R., Rosm A. nd Drh, I. 2010. Inhitory effect of lctic cid nd nisin on cteril nd spoilge of chilled shrimp. World Acdemy of Science Engineering nd Technology, 65, 163-167. Szo, E.A. nd Chill, M.E., 1998. The comined effects of modified tmosphere, temperture, nisin nd lt (Tm) 2341 on the growth of Listeri Monocytogenes. Interntionl Journl of Food Microiology, 43, 21-31. Teerkrn, A., Hirt, D.E., Acton, J.C., Rieck, J.R. nd Dwson, P.L., 2002. Nisin diffusion in protein films: effects of film type nd temperture. Journl Food Sciences, 67, 3019-3025. Thoms, L.V. nd Delves-Broughton, J., 2005. Nisin. In ntimicroils in food,, 3rd ed., Dvidson, P.M.S., J. N., Brnen, A. L (Ed.) CRC Press Tylor & Frncis Group, LLC: Boc Rton, FL, pp. 237-273. Torrieri, E., Cvell, S., Villni, F. nd Msi, P., 2006. Influence of modified tmosphere pckging on the chilled shelf life of gutted frmed ss (Dicentrrchus lrx). Journl of Food Engineering, 77, 1078-1086. Wolf, C.E. nd Gions, W.R., 1996. Improved method for quntifiction of the cteriocin nisin. Journl of Applied Bcteriology, 80, 453-457. Xio, D. nd Zhong, Q., 2011. In vitro relese kinetics of nisin s ffected y tween 20 nd glycerol co-encpsulted

Irnin Journl of Fisheries Sciences 14(2) 2015 381 in spry-dried zein cpsules. Journl of food engineering, 42(1), 75-83. Xio, D., 2010. Novel delivery systems of nisin to enhnce longterm efficcy ginst foodorne pthogen listeri monocytogenes. Doctorl Disserttions. University of Tennessee, Knoxville. pp. 1-3. Zhong, Q. nd Jin, M., 2009. Nnosclr structure of spry-dried zein microcpsules nd in vitro relese kinetics of the encpsulted lysozyme s ffected y formultions. Journl of Agriculturl nd Food Chemistry, 57, 3886-3894. Zhu, M., Du, M., Cordry, J. nd Ahn, D.U., 2005. Control of Listeri monocytogenes contmintion in redy-to-et met products. Comprehensive Reviews in Food Science nd Food Sfety, 22, 34-42.