Life Science Journal 2015;12(2)

Comintions of nisin n γ irrition to improve miroiologil qulity of mine hiken uring refrigerte storge Dli A. Zhrn Helth Rition Reserh Dept., Ntionl Centre for Rition Reserh n Tehnology (NCRRT), Egyptin Atomi Energy Authority, Egypt slmr_ysser@yhoo.om Astrt: This stuy evlute the effet of nisin t supplementtion levels of 100 or 200 µg/g, γ irrition t ose of 1 or 2 kgy n their omintion, on miroiologil qulity of mine hiken n mine hiken rtifiilly inoulte with Billus ereus n Esherihi oli, uring refrigerte storge. Smples were nlyze for totl teril, mol & yest ounts t 0-time, 7 n 14 ys n the log numer of miroorgnisms were etermine. Tretment of mine hiken with nisin lone t 100 or 200 µg/g showe no signifint (p> 0.05) improvement in miroiologil qulity of mine hiken uring storge. The omintion of nisin n γ irrition improve (p< 0.05) the miroiologil qulity of mine hiken together with the ntiteril tivity ginst Billus ereus n Esherihi oli. The use of nisin omine with γ irrition woul e promising to ontrol foo-orne pthogens, improve miroiologil sfety n exten shelf-life. [Dli A. Zhrn. Comintions of nisin n γ irrition to improve miroiologil qulity of mine hiken uring refrigerte storge. Life Si J 2015;12(2):147-152]. (ISSN:1097-8135). http://www.lifesienesite.om. 21 Keywors: Mine hiken, nisin, γ irrition n miroiologil qulity. 1. Introution Miroorgnisms ontrol in met prouts is the mjor onern in the preprtion of high qulity foos. Fresh hiken n hiken prouts n e esily ontminte with miroorgnisms n, if not properly hnle n preserve, support growth of spoilge n pthogeni teri, leing to loss of qulity n potentil puli helth prolems (Vernozy-Rozn et l., 2002). Spoilge of fresh hiken prouts is n eonomi uren to the prouer, onsequently eveloping methos to prolong the shelf-life n overll sfety/qulity represents mjor tsk of the hiken inustry. A reent tren in foo proessing reserh hs fouse on meeting onsumer emns for more nturl n fresher foo. The pplition of omintions of ifferent non-therml hurles, use t su-lethl levels, oul mintin the orgnolepti n nutritionl qulity while still ensuring the sfety n stility of the foo prout (Leistner n Gorris, 1995). One metho s mens to non-thermlly intivte pthogens in foo prouts is gmm (γ) irrition. Irrition offers gret potentil for pplition in foo preservtion (Frks, 1998, Frks, 2006). New ttempts in foo irrition tehnology onsist to omine rition tretment with other gents re of utmost importne in enhning the miroiologil effetiveness, reuing the energy requirement for foo preservtion n improving prout qulity (Frks, 1989). It hs een previously emonstrte tht the irrition in presene of vrious nturl or syntheti tive ompouns woul help to inrese the riosensitiztion of foo pthogens (Lroix n Outtr, 2000). Riosensitiztion is well-known iologil phenomenon whih my e of prtil utility in enhning the tehnil effetiveness n fesiility of irrition. The spetrum of pplitions of foo irrition oul e expne in omintion with terioins, (Glvez et l. 2007). The empiril use of miroorgnisms n/ or their nturl prouts for preservtion of foos (iopreservtion) hs een ommon prtie in the history of mn kin (Ross et l., 2002). Nisin, the only terioin with foo n rug ministrtion (FDA) pprove GRAS (Generlly Regre As Sfe; US 21 CFR170.30-Foo Aitives), is suessfully use s ntiteril gent in vrious foo prouts (Jy, 2000). Nisin is more effetive ginst spores thn ginst vegettive ells (Bell n ely, 1985). It is effetive ginst Grm positive teri ut oes not inhiit the mjority of Grm-negtive teri. Nisin ts minly y permeting the ytoplsmi memrne with the formtion of trnsient pores. The mehnism of tion of this polypeptie terioin involves ining to the peptioglyn lyer, using estiliztion of the memrne y formtion of trnsient pores, whih llow lekge of intrellulr metolites n issiption of memrne potentil (interferene with memrne funtion) (Bruno et l., 1992). Aoring to Bue n Rsh (2001), the pore formtion o not neessrily le to ell eth s ells my repir when trnsferre to rih growth mei without terioin.. Nisin is stle uner 147

refrigerte onitions, emonstrtes het stility, ut is egre y gut enzymes. By now, the ntimiroil tivity of vrious omintions of nisin with γ irrition in mine hiken hs not een exmine. Therefore the im of this work ws to evlute the inhiitory effet of nisin pplie singly or in omintion with γ irrition to improve the miroiologil qulity of mine hiken n the ntimiroil effet ginst rtifiilly inoulte Billus ereus n Esherihi oli in mine hiken uring refrigerte storge. 2. Mterils n Methos 2.1. Nisin preprtion Pure nisin with lel tivity of 10 6 IU/g ws provie y Aplin n Brrelt Lt. Trorige, UK. Nisin ws soluilise in 0.02 M HCl t onentrtion of 10 mg/ml with heting (60-70 o C) to i soluilistion. The solution ws sterilize y filtrtion through 0.22 µm memrne filters prior to use. 2.2. Preprtion of mine hiken n tretments Chiken rsses were purhse from lol poultry proessing plnt immeitely fter evisertion. They were trnsporte to the lortory in n ie ox n store t 4 o C for no longer thn 2h efore use. Met ws seprte from the ones n mine in n eletril miner. Ten grm smples were septilly sele in polyethylene gs fter the tretment with ifferent nisin onentrtion n/ or γ irrition oring to the sheme expline in Tle 1. Pkges were then store on the refrigertor (4± 1 o C) shelf until nlysis. Three pkges from eh group were exmine every nlysis. 2.3. Irrition proess: Pkge mine hiken smples were irrite oring to the sheme in Tle 1 with 0, 1 n 2 kgy gmm irrition t ose rte 3.49 kgy/ h using the Inin Gmm Chmer 4000 A with 60 Co soure. The irrition proess ws onute t the Ntionl Center for Rition Reserh n Tehnology (NCRRT), Nsr ity, Ciro, Egypt. After irrition, ll smples were kept in refrigertor t 4± 1 o C until exmintion. Three pkges from eh group were nlyze immeitely fter irrition (0- time), 7 n 14 ys. 2.4. Miroiologil nlysis: For etermintion of miroil ounts, 10 g of eh smple were homogenize in 90 ml sterile physiologil sline solution (0.85% NCl), then seril 10-fol ilutions were prepre. Approprite seril ilutions were uplite plte (spre metho) with plte ount gr meium for totl teril ount (TBC) n zpek yest extrt gr meium for mol & yest ounts (M&YC). Pltes were inute t 37 o C for 48 h for TBC n 25 o C for 5 ys for M&YC (APHA 2001). 2.5. Bteril strins n inoultion: Lol strin of Billus ereus n Esherihi oli (previously isolte n ientifie in the Foo Hygiene Dept., Animl Helth Reserh Institute, Dokki, Egypt) mintine on nutrient gr (Oxoi) were use to rtifiilly ontminte mine hiken smples. Eh orgnism ws grown seprtely in 50 ml nutrient roth (Oxoi) roth for 24 h t 37 o C. Then the ultures were serilly ilute in sterile sline (0.85% NCl) for stnriztion y pour plte ssy in uplite using nutrient gr pltes inute t 35 o C for 18 h. A finl Billus ereus n Esherihi oli inoulum of 10 6 n 10 8 CFU/ ml, respetively were use seprtely for the inoultion of smples. Smples use for rtifiil inoultion test were first sterilize using 20 kgy y elerte eletrons in the NCRRT (energy: 1.5 Mev n urrent: 0.9 m A). One ml of the inoulum of eh orgnism ws inoulte iniviully in smple (3 pkges for eh tretment for eh orgnism) fter the onfirmtion of steriliztion test y plting on plte ount gr (PCA) meium. Survivors of Billus ereus n Esherihi oli were enumerte on PCA using spre tehnique fter inution t 35 o C for 24 h. 2.6. Sttistil nlysis: Mine hiken smples from 9 (miroiologil nlysis) n 5 (ntimiroil effet) ifferent groups were nlyze. Miroil popultions were onverte to log 10 CFU/g. The vlues given in eh tretment tegory re the men vlues of three iniviul smples. Anlysis of vrine (F-test) ws one for smples in eh storge perio using the Generl Liner Moel (SAS, 2002). Lest signifint ifferene (LSD) (P< 0.05) ws performe on the exmine orgnisms. 3. Results 3.1 Miroiologil nlysis The inhiitory effets of nisin t 100 or 200 µg/g, γ irrition t 1 or 2 kgy n their omintion on totl teril (TBC) n mol & yest (M&YC) ounts in mine hiken smples store t refrigerte (4± 1 o C) temperture for 14 ys re shown in Figs. 1 n 2, respetively. Aition of nisin to mine hiken smples t 100 or 200 µg/g (groups B n C) resulte in TBC (Fig. 1) n M&YC ounts (Fig. 2) not signifintly (p> 0.05) ifferent thn those of the ontrol smples neither t 0-time nor t the en of storge, initing no ntimiroil tivity of nisin lone t this level. Using γ irrition t 1 or 2 kgy (group D n G) signifintly (p<0.05) reue oth TBC (rnging from 1 to 1.4 log CFU/ g) n M&YC (rnging from 0.89 to unetete level) ompre 148

with groups A, B n C t 0-time. While t the en of storge, this reution rnge from 1.86 to 2.99 n from 1.81 to 3.32 log CFU/ g, respetively. The omintion tretment of nisin (100 or 200 µg/g) n γ irrition (1 kgy) (group E n F) resulte in erese in the TBC n M&YC ut this erese ws non signifint (p> 0.05) ompre with group D (1 kgy γ irrition only) throughout ll storge perio exept M&YC for group F t the en of storge. The sme tren ws foun in M&YC for group H n I ompre with group G. On the ontrry, there ws signifint (p< 0.05) reution in TBC of group I ompre with group G throughout ll storge perio. At the en of storge, TBC n M&YC of the ontrol (group A) rehe 7.83 n 5.55 log CFU/ g, respetively. While these ounts rehe 3.92 n 1.68 log CFU/g, respetively, in group I. Although there ws 0.55 log CFU/ g reution in M&YC in smples trete with omintion of 2 kgy n 200 µg/g (group I) ompre to those trete with 2 kgy lone (group G), ut this reution ws non signifint (p> 0.05). 3.2 Antimiroil tivity in rtifiilly inoulte mine hiken The effets of omintion tretments of nisin (100 or 200 µg/g) n γ irrition (1 or 2 kgy) on Billus ereus n Esherihi oli in mine hiken smples store t refrigerte (4± 1 o C) temperture for 14 ys re shown in Figs. 3 n 4, respetively. Due to psyhotropi nture, the initil popultion of Billus ereus (5.35 log CFU/ g) in ontrol smples inrese rehing 8.12 log CFU/ g y the en of storge. It ws ler tht there ws signifint (p< 0.05) reution in the survivors of Billus ereus (Fig. 3) in ll omintion tretments ompre with the ontrol throughout storge perio. Although there ws non signifint (p> 0.05) erese in numer of Billus ereus survivors in group H n I t 0-time n fter 7 ys of storge, ut this erese ws signifint (p< 0.05) t the en of storge. At the en of storge (fter 14 ys), Billus ereus ws unetete in group I. Conerning rtifiilly inoulte Esherihi oli, it ws ler from Fig.4 tht there ws highly signifint (p< 0.05) reution in the numer of survivors in ll tretments throughout storge perio ompre with the ontrol. Esherihi oli ws unetete in group H t the en of storge n in group I fter 7 ys initing its sensitivity to the omintion tretments uner investigtion. Tle 1. Explntion of group nmes, nisin onentrtion n irrition ose. Irrition ose Nisin (µg/g) (kgy) 0 100 200 0 A B C 1 D E F 2 G H I Figure 1 Totl teril ount of mine hiken (n=3) trete with (A) 0 kgy γ irrition +0 µg/g nisin, (B) 0 kgy γ irrition + 100 µg/g nisin, (C) 0 kgy γ irrition + 200 µg / g nisin (D) 1 kgy γ irrition + 0 µg/g nisin, (E) 1 kgy γ irrition + 100 µg/g nisin, (F) 1 kgy γ irrition + 200 µg / g nisin, (G) 2 kgy γ irrition + 0 µg/g nisin, (H) 2 kgy γ irrition + 100 µg/g nisin n (I) 2 kgy γ irrition + 200 µg / g nisin uring storge. Figure 2 Mol & yest ount of mine hiken (n=3) trete with (A) 0 kgy γ irrition + 0 µg/g nisin, (B) 0 kgy γ irrition + 100 µg/g nisin, (C) 0 kgy γ irrition + 200 µg/g nisin (D) 1 kgy γ irrition + 0 µg/g nisin, (E) 1 kgy γ irrition + 100 µg/g nisin, (F) 1 kgy γ irrition + 200 µg/g nisin, (G) 2 kgy γ irrition + 0 µg/g nisin, (H) 2 kgy γ irrition + 100 µg/g nisin n (I) 2 kgy γ irrition + 200 µg/g nisin, uring storge. 149

Log num er (C FU/ g) 9 8 7 6 5 4 3 2 1 0 0 7 14 Storge ( ys) Figure 3 Artifiilly inoulte Billus ereus survivors of mine hiken (n=3) trete with (A) 0 kgy γ irrition + 0 µg/g nisin, (E) 1 kgy γ irrition + 100 µg/g nisin, (F) 1 kgy γ irrition + 200 µg/g nisin, (H) 2 kgy γ irrition + 100 µg/g nisin n (I) 2 kgy γ irrition + 200 µg/g nisin, uring storge. Log num er (C FU/ g) 8 7 6 5 4 3 2 1 0 0 7 14 Storge (ys) Figure 4 Artifiilly inoulte Esherihi oli survivors of mine hiken (n=3) trete with (A) 0 kgy γ irrition + 0 µg/g nisin, (E) 1 kgy γ irrition + 100 µg/g nisin, (F) 1 kgy γ irrition + 200 µg/g nisin, (H) 2 kgy γ irrition + 100 µg/g nisin n (I) 2 kgy γ irrition + 200 µg/g nisin, uring storge. 4. Disussion The results of this pper lerly emonstrte the effetiveness of using omintion of nisin n γ irrition ginst TBC, M&YC, Billus ereus n Esherihi oli. At 0-time, TBC of the ontrol (group A) ws 5.23 log CFU/g initing nee for improving miroiologil qulity of mine hiken y using some preservtives tht oul e esily e A E F H I A E F H I pplie in oth lrge n smll sle proutions. After 14 ys of storge, TBC of the ontrol (group A) rehe 7.83 log CFU/ g whih ws higher thn the Egyptin stnrs (4178/ 2005) n shoul e rejete. The TBC is onsiere n inex of qulity whih gives n ie out the hygieni mesures uring proessing n helps in the etermintion of the keeping qulity of the prout (Aerle et l., 2001). High TBC might e ttriute to the ontmintion of the prout from ifferent soures or unstisftory proessing s well s it my e ue to unsuitle onition uring storge (Cox et l., 1998, ICMSF, 1980). Mol n yest ount of group A ws 2.92 log CFU/g t 0-time whih inrese to reh 5.55 log CFU/g fter 14 ys. Mol n yest re use s inex of snittion n their high ounts elerte spoilge. Yest my grow uner eroi onition on the surfe of mets using sliminess, lipolysis n pink to rown olortion ue to the pigments in the yest. Mol n ssist in the putreftive proesses, they n grow over wie rnge of temperture n my use moly oor n tste to foo, they my lso proue myotoxins whih re hrmful to mn n niml (Frzier n Westhoff, 1983). The ition of nisin t 100 or 200 µg/g to mine hiken i not use ny inhiitory (p< 0.05) effet on TBC n M&YC uring refrigerte storge ompre to the ontrol. This fining ws similr to tht reporte y Broz De Mrtinez et l. (2002). Chung et l. (1989) foun tht nisin n ely the growth of Grm-positive teri tht re tthe to met. However, nisin lone my not e suffiient to prevent spoilge, sine grm-negtive n nisinresistnt grm-positive teri suh s lti i teri re often ssoite with met spoilge. It is lso known tht the ntiteril tivity of nisin is wekene in mine met ue to its ining to proteins n ft, or its retion with met proteses (Asen et l., 2003, Stergiou et l., 2006). The use of γ irrition t 1 or 2 kgy resulte in signifint (p< 0.05) reution in TBC n M&YC ompre with the ontrol. The immeite reution fter γ irrition my e minly ue to the iret effet of gmm rys on the miroil ell y using lesions in the geneti mteril of the ell, effetively preventing it from rrying out the iologil proesses neessry for its ontinue existene (Murno, 1995). He e tht miroorgnisms whih re responsile for spoilge of re met, s well s those tht re pthogeni, re esily estroye y the ose use in ol psteuriztion (1 to 10 kgy). Generlly, it is known tht Grm-negtive teri whih inlue mny spoilge n pthogeni orgnisms re quite sensitive to rition. The spoilge flor of met is usully ominte y the 150

teri whih grow most rpily uner the storge onitions of the prout. Uner eroi onitions, the ominnt spoilge orgnisms re stritly Pseuomons spp. (Kntt et l., 2005). The sensitivity of these orgnisms to rition explins the signifint eline (P< 0.05) in their ounts. By refrigerte storge, the miroil ounts were inrese progressively (P< 0.05). This fining gree with tht reporte y Blmtsi et l. (2006) in hiken met. Giroux et l. (2001) mentione tht gmm irrition lowere the rte of inrese in the numer of miroorgnisms in irrite eef ptties thn tht in non-irrite ontrol. Billus ereus is spore forming, Grm positive, eroi, ros teri. It hs een long known s uiquitous orgnism foun in ir, soil n wter (Clus n Berkeley, 1986). Billus ereus is the etiologi gent of two istint types of foo poisoning (Thyer n Boy, 1994). They lso stte tht ontrolling sttionry phse ells n enospores my e more importnt for foo sfety thn ontrolling logrithmi phse ells euse of the high rition resistne of the enospore n euse the sttionry phse woul proly e rehe in most use foos. In stuy one y Zhrn et l. (2008), the iniene of Billus ereus in mine hiken ws 60% of exmine smples. Presene of E. oli in foo prouts inite iret or iniret ontmintion of fel origin or might e ue to hnling uner unhygieni onitions (Frzier n Westhoff, 1983). Severl uthors (Cleveln et l. 2001, Solomkos et l., 2008) reporte tht nisin i not possess ntimiroil tivity ginst Grm-negtive Esherihi oli in mine eef. This might e ue to inility of nisin to ttk Grm-negtive teri, ue to the protetive role of the outer memrne, whih overs the ytoplsmi memrne n peptioglyn lyer of Grm-negtive ells (Fng n Tsi, 2003). The 3 kgy gmm irrition h signifint (P<0.05) effet on elimintion of E.oli (Jo et l. 2005). The response of teri to irrition in ifferent environments hs shown tht vrious foo mtries n offer ifferent egrees of protetion to the orgnism, lso ifferenes in reovery tehniques (Diehl, 1995). Sine irrition ts on DNA (Urin, 1986) n nisin ts on the teril ytoplsmi memrne (Bruno et l., 1992), the effetiveness (p< 0.05) of the omintion of nisin (100 or 200 µg/g) n γ irrition (1 or 2 kgy) in reuing TBC, M &YC, Billus ereus n Esherihi oli in the present stuy ws not surprising. The enhne ntimiroil tivity ws expline y severl investigtors (Rike et l., 2005, Glvez et l., 2007) tht the omintion of more thn one intervention tretment hs een foun to proue greter ntimiroil effet thn ny single tretment (whih is referre to s hurle tehnology), often working synergistilly, with lower oses ompre to their iniviul pplition n very low proilities for survivl n prolifertion for ells onfronte with multiple hurles. Ayri et l. (2012) oserve tht the inhiitory effet of γ irrition (1 kgy) inrese signifintly (p< 0.05) in presene of nisin. The pre-tretment of teril ells with nisin, proue greter log reution thn the only irrite ontrol, s it renere ells more suseptile to the su-lethl ionizing rition t 1 kgy. 5. Conlusion The use of nturl itives in foo represents itionl hurle tehnologies tht my omplement or t synergistilly with tritionl intervention pprohes. In ition, some of these nturl ompouns my exhiit ntimiroil properties tht iffer mehnistilly from other ntimiroils eing use. Tretment of mine hiken with nisin t 100 or 200 µg/g i not show ny ntimiroil tivity ginst TBC n M&YC. The omintion of nisin with γ irrition showe n itionl effet (p< 0.05) ginst TBC n M&YC uring refrigerte storge. It lso h signifint (p< 0.05) ntimiroil tivity ginst Billus ereus n Esherihi oli in mine hiken. Finlly, this omintion shoul not e use s sustitute for omplying with generl hygieni prties when hnling n prepring foo. Referenes 1. Asen I.M., Mrkussen S., Moretro T., Ktl T., Axelsson L. & Nterst K. 2003. Intertions of the terioins skin P n nisin with foo onstituents. Int Foo Miroiology, 87, 35-43. 2. Aerle E.D., Forrest J., Gerrr D.E. & Mills E.W. 2001. Priniples of Met Siene (4 th e). Hunt Pulishing Co., Kenll, USA. 3. APHA 2001. Compenium of methos for the miroiologil exmintion (4th e.). Wshington: Amerin Puli Helth Assoition. 4. Ayri S., Dussult D., Hyouni E., Vu K.D., Hmi M. & Lroix M. 2012. Response of Billus ereus vegettive ells fter exposure to repetitive sulethl rition proessing in omintion with nisin. Foo Miroiol, 32, 361-370. 5. Blmtsi C.C., Rogg K., Bek A., Kontomins M.G. & Svviis I.N. 2006. Effet of low ose rition on miroiologil, hemil n sensory hrteristis of hiken met store eroilly t 4 egrees C. Foo Prot, 69, 1126-1133. 6. Broz De Mrtinez Y., Ferrer K. & Sls E.M. 2002. Comine effets of lti i n nisin solution in reuing levels of miroiologil ontmintion in re met rsses. Foo Prot, 65 (11), 1780-1783. 7. Bell R.G. & ely K. M. 1985. The effet of nisinsoium hlorie intertion on the outgrowth of 151

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