Identification Characteristics of Irradiated Dried Red Pepper during Storage by Analysis of Thermoluminescence, DNA Comet, and DEFT/APC

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 6, pp. 851~856 (2004). ©The Korean Society of Food Science and Technology. Thermoluminescence, DNA Comet 5 DEFT/APC ª+ö ~ OÒ.¾Ò º~ &Ë 7 ¦æ ß9 B÷Á²%^* ã§v ®". Identification Characteristics of Irradiated Dried Red Pepper during Storage by Analysis of Thermoluminescence, DNA Comet, and DEFT/APC Byeong-Keun Kim and Joong-Ho Kwon* Department of Food Science and Technology, Kyungpook National University Minerals separated from irradiated dried red pepper (whole) at 2.5 kGy or higher showed typical thermoluminescence (TL) glow curves (TL1) at around 150oC, which increased with irradiation dose. The TL ratio (TL1/TL2) through re-irradiation step at 1 kGy enhanced reliability of TL identification results. DNA comet assay indicated that the intact cell was observed in non-irradiated pepper (seed), while some long tails were found in irradiated ones, showing relationship between irradiation dose and tail length. Log DEFT/APC values increased in proportion to irradiation doses in powdered and whole peppers. Based on overall results, irradiated dried red peppers could be screened using DNA comet assay or log DEFT/APC, and moreover the identification results were verified by TL analysis. Key words: red pepper, irradiation, thermoluminescence, DNA comet assay, DEFT/APC. *. . ~ Ï Bf ¦&Ò GöB ^B6b æ'> ®. (6,7). V¢B Ï Bj Oæ~V *Bº zîÃB~ ÒÏ ¢>' O»îb¾ n*W 5 ~ã GöB îÚ &n~ BB B'b º> ® (6). *Ò OÒFf ßF b' ·Ïb Ï 5 bj Ò Ò > ®b(2,7), *Ò «~~ ® 5 ® öòö ' Ï î&>Ú ® (8). ÖÒ¾¢~ "º º >Â 7f çëÏ OÒF ÒJj <º ®bæ ¢¦ ³Öbö &~ OÒF ¾Ò ¢ ¯~º ©b rJê (4,8). V¢B v *~ &Ò 5 ®î ;j :ûb n*&Òf ß® >« ê OÒF ÒÒ(re-irradiation)¢ Ò*ö Oæ~V *Bº OÒF ¾Ò ®~ Î"' {VF(identification methods)~ {& /® º> ® (4,7,9). OÒF Ò®~ {/¦æ VFf ² bÒ' O», b' O», z' O»(9)b ª > ®b, *Ò Ï &ËW {> ®º O»bº b« Jª~ B7 ß Wj Ï thermoluminescence(TL) ªC(9-11), Jªö & 7¶ öÒ¢ Ï photostimulated luminescence(PSL) ªC (12,13), free radical ³ê¢ G;~º electron spin resonance (ESR) ªC(9,14), Ò GC $º GC-MS¢ Ï æOî F ¾~ hydrocarbon~ 5 2-alkylcyclobutanones~ ªC(9,15), DNA ¶ç" "V æz¢ {~º DNA comet assay(16,17),. ®~ OÒF ÒVFf n*W" VF' æW BV(FAO/IAEA/WHO)ö ~ 'b ;>Ú B B" Ï ²Ë> ® . ¯, Codex ®Ï*ö²öBº OÒF Ò ® 5 ¾Ò Jö & CodexÏ 5 Ú' ;j j~ ' ö Ïj ²Ë~ ® (1,2). *Ò ^ê 'b OÒF Ò& î&B ®~º 230 «b &¦ª ~ ® >Ú ® (3). 7 çë' Î OÒF ¾Ò> ®º ®~º &¦ª Ëò, ~, ®~, G ~, "¢~, j²~ , 7 Ëòf ò¢ ®~~ î& 5 Ïz& &Ë B~ (4). ÖÒ¾¢~ &' Ëò º~ ãÖöº &¦ª &>Ú ²j> ® . Öïf 2000j" 2001jö 5~ >&b Ã&Nj &æò, ³Öb Ë~ BOb vï ~ Ã&f z®Ú º~ >«ïf >Âïö j Ã&~º º^ (5). ºº &Ë 7 Ï& B>, vç®öB *Corresponding author: Joong-Ho Kwon, Department of Food Science and Technology, Kyungpook National University, Daegu 702701, Korea Tel: 82-53-950-5775 Fax: 82-53-950-6772 E-mail: [email protected] 851.

(2) ®"²æ B 36 ² B 6 ^ (2004). 852. .V b >f Ò ê b >~ æz¢ G;~º DEFT/ APC O»(18) B® > ®b¾, OÒF ¾ÒB º~ ¦*ê ¦æ º jç êB : ì . öBº º 5 ªö~ Î"' { O» ¢ *~ bÒ' ªC» TL" b' O» DNA comet assay 5 DEFT/APC O»j Ï~ º ¦*ê radiationinduced marker ~ {" ~ &Ë 7 ªC &ËWj ¦ Æ~& .. Òò 5 O» Òò þö ÒÏB º(Capsicum annum L.) òº 2002 j ÖæöB «~&b º O}*öB ªöz(powder) ©" Ûº(whole)¢ ÒÏ~ þj ~&b, DNA comet assay~ ãÖöº Ûº(whole)öB N(seed)¢ ªÒ~ ÒÏ~& . 6î. Ò Òö2 ÏVö ËB º ò~ 6îF Òº 60Co 6îF ÒJ(AECL, IR-79, MDS Nordion International Co. Ltd., Ottawa, ON, Canada)j Ï~ B'b îÏB Fï º* 0-10 kGy~ C >Fïj áê ~& . r ceric/ cerous dosimeter¢ Ï~ >Fï~ {j ~& (Û 5%). ÒB òº N7B Nö &Ë~B þö ÒÏ~& . Thermoluminescence (TL) ª+ OÒF Ò ò~ TL ªCf CEN(19) O»ö &~. ~&b, TL G;j * jº Jªj áV * .j. þj ~& . Ö" Ûº~ ãÖ £ 600 g~ ·j water rinsing ~º O»b Jªj j > ®î . ¯, Ûº ö ¢;ï~ Ã~>¢ &~ ultrasonic agitator(Branson 3210, Branson Ultrasonic Co., Danbury, CT, USA)öB 5ª* ¾Ò ê ò¢ water rinsing~, 125 µm sieve¢ Û" * ¢;* ;~Î ê *bj ~& . *bf sodium polytungstate solution(2.0/mL) 5 mLj &~ FVbj B~ Ã~> ^¿~& . ¢>'b b«B Jªöº carbonate ¢ F~ ®b carbonateº ÒÒ ê G; 1N G ;öB~ glow curve peakö & 'Ëj .¾~æ 1 N HCl 2 mL¢ & 10ª* z²öB ;~~ cabonate¢ B~& 1 N NH4OH 2 mL¢ & 7z 8 . 7zB Jªf Ã~ > Ïª® ^¿ ê acetoneb Nf ^¿ ê 8 . B Jª 1 mgf aluminium disc(φ6 mm)ö TÎ 50oC incubatoröB ~!J ;ê . ê TLD system (Harshaw TLD-4200, Dreieich, Germany)j Ï~ high pure N2 gas(99.99999%)¢ ~ ÚB G;~& . TLD~ ªC f .V Nê¢ 50oC ~ 5. ÿn . ê N ê 400oC, &NNf 50oC/sec ~&b, acquisition timej 70 . ~ G;~& . G;B ò~ TL ßWj j*® B ~V* 5. ÿn~ annealing timej J;~& . OÒF Ò ¦~ {f G;B glow curve¢ TL intensity(TL1) ¾ æÚîb, TL1~ normalizationj *~ G;B Jªö Ò Ò(1 kGy)¢ ~ 2N glow curve(TL2)¢ G;~& . ê TL1/TL2~ 'j¢ ~ Ò ¦ {~ ÖWj ¸ V * threshold value¢ ÖÂ~& (20).. DNA comet /; º~ comet assayº N¢ Fê~ ~&b, single cell gel electrophoresis(17)ö V¢ 0.5% agarose(normal melting point agarose, Sigma) Ïj Ï~ pre-coated slide¢ &j ~, ¦#² îêB 1.5 g~ òö ïË&(4oC)B PBS(phosphate buffe red saline, pH 7.4)¢ 5 mL &~ stirring plate¢ Ï~ îz Ê mess 200 µm, 125 µm nylon sieve cloth¢ Nf Û"* ºÒ¢ B~& . " B ^ *çf 20ª ÿn, 4oC~ ïËö O~Î ê ç [j þö ÒÏ~& . Gel castingf ^*ç 50 µL f 0.8% low melting point agarose(LMA) 100 µL¢ b~, cover glass¢ Ï~ ¢~² ê ê ice bath *ö 10ª * O~~ gelj ;W8 . Casted slidesº lysis buffer(2.5% SDS in 45 mM tris-borate, 1 mM EDTA, pH 8.4)ö 35ª ÿn (21) æ ê Ã~> ^¿~& . LysisB slideº SDS& V BB TBE buffer(Tris borate electrophoresis buffer, pH 8.4)ö 5ª ÿn æ~, *V'ÿ > *ö agarose end& anodeO Ëb Ë~ê ¹f ê TBE buffer& Z 2 V/cm ;{~ ² 2ª* *V'ÿj ~ Ã~> ^¿ ê ~&. . Î slide glassº ethidium bromide(EtBr) ;7"ï £ bathö 20ª ÿn æ~ "ï ê ^¿~& . ^¿B slide glassº pasteur pipetteb Ã~>¢ OÞ ÎÚNÖ ê cover glass¢ 2Ö 595 nm emission filter& ËOB Laser Scanning Confocal Imaging System MRC-1000(Biorad, Hercules, CA, USA)¢ Ï~ 20V VN &V~& . &VB DNA tail migrationf Laser Sharp software(Biorad, Hercules, CA, USA)¢ Ï~ Ú 7öB tail ræ~ ^¢ tail length ~ G;~&b, r Î òö & 2B~ slide ¢ &j~ ' slide DNA 25BO C 50B~ Ú¢ &ç b G;~& . DEFT/APC /; ò~ DEFT/APC G;(18)öB aerobic plate count(APC)º >¢ G;~º O»b 10V C»b CB ò Ïj '' ¢;ïj ~ ï6Væ(plate count agar, Difco) ö êö ~ 30Û1oC NV·VöB 72* ÿn Ê÷ÚB V·~& . V·B ^V' >º counting~ C»b êÖ r log unit ~Ö~& . DEFTº > 5 j >~ C>¢ G;~º O»b 10V C»b C B CÏ 2 mLOj ê" Ë~& ¦O>Ú ®º "V (filtration tower, Millipore) Tâ . "Vº stainless steel >Ú ® bottom filter~ çã 25 mm, Ò tower~ ¦b º 30 mL& . Filter paperº prefilter¢ * polypropylene filter(çã 25 mm, pore size 10 µm, German Science Inc.)f membrane filter¢ * white polycarbonate filter(çã 25 mm, pore size 0.6 µm, Nuclepore)¢ ÒÏ~& . " 2 mLº membrane filter ÿn ê 25 mmHg¢ >æ p 20 mmHg ¢ Fæ~ê ~& . "B membrane filter paperº C> G;j * acridine orange(25 mg/100 mL buffer, pH 6.6) "ï~ paper¢ pH 3.0 bufferf isopropanol 2.5 mL ^¿~& . ^¿B polycarbonate filter paperº Ê#² slide glass*ö ¹ V Î r immersion oilj ÎÚN J paper¢ ' cover slipj f ê ªCö ÒÏ~& . & j slide~ &Vf ;7*ã(Carl Zeiss Axioplanz Imaging, Gotengen, Germany)b 100VN ;~ G;~& . &V.

(3) OÒF" îÃ ¾ÒB º~ ¦æ ßW. 853. B C>f >~ jº Wirtanen" Sj berg(28)~ O»ö V¢ êÖ~& . ¯ ò g DEFT(X)º *ã &V ' (microscope field, N/n), ò~ CV>(DF), microscopy factor (MF) DEFT unit~ ï8b êÖ~& . f r" ? . X = DEFT count/g = (NÜMFÜDF)/n VöB Nf n microscope fieldöB êÖB DEFT units~ , nf G; microscope field~ >, MFº microscopy factor(=FA/MAÜV) . FAº membrane filter~ ' (πR2, Rf filtration tower~ 7 ¦ª >ã) MAº microscope field~ '(πR2, rf microscope field~ >ã) Vº ò~ ¦b(mL) . DEFT/APC~ log unit 8f ^ ® > þ Ö" êÖ r ï8j ~& . Fig. 1. Typical glowcurves of minerals separated from irradiated whole red pepper.. Ö 5 V OÒ. Ò º~ TL ß9 TL signal G;j * *¾Ò ";öB .j þj Û 600 g~ ò¢ water rinsing~ 0.1 mg~ Jªj ºÂ~ TLö ÒÏ~& . r þ~ Öê¢ ¸V * Jª ~ ªÒ ·öBº Jª ò& Îææ pf þ&öB ê ?f ·~ :ûþj ~ *¾Ò 7 Jª J" ¦¢ {~& . Ö" 4.780-13.78 nC º*öB TL glow curve& G;>îb, º jÒf FÒ ;ê~ ' 8 j B þ 7ö Jª~ F«¾ J" ¢Ú¾æ p f ©j {~& . º¢ Ëò~ ãÖ Ú î& Fï 10 kGyæ öBº 2.5 kGy¢ & Fïb ;~ þ~& . Fig. 1ö ¾æÞ :f ? 2.5 kGy ç Ò òöBº ß' peak& 150oC *êöB ¾æÒ, ÒFï~ Ã&ö V¢ glow curve(TL1) V& Ã &j {~& (R2 = 0.9540). ¾ jÒ òöBº ? f Nê º*öB glow curve¢ { > ìî . ?f Ö "º «~~ OÒF Ò ®öB B B7 ßW " FÒ~&b(4,9-11), ò*~ signal intensity¢ jvb jÒf Ò*~ ê &Ë~& . $ OÒF Ò òöB ¾æÂ TL1 glow curve¢ ¦Ã~V *~ normalizationj Û TL ratio(TL1/TL2 'j)¢ ÖÂ Ö", j Òº 0.001, 2.5 kGy ç Òº 0.814 ç~ ¸f 8 j TL1 ªC Ö"~ Öê¢ ¸¢ > ®î (19-24). $ NöB 8Bú ÿn &Ë º¢ &çb TL signal j G;~&j r ÒFï" F~' ç&&ê(R2 = 0.8741) ¢ Fæ~&(Table 1) TL ratioöBê &Ë .Vf FÒ ã Ëj & . º «~~ herbf spice¢ &çb whole sample testö ~ TL G;öB Ò ê 9Bú ræê OÒF Ò ¦~ 6ê &Ë~ Mamoon (25)~ Ö"f FÒ~& .. OÒ. Ò º~ DNA comet ß9 OÒF ÒB º~ DNA cometj &V~V *B " bf Nö & .j þj ~&j r "b~ ãÖöº DNA comet &V>æ p~ . ¾ N~ ãÖöº Khan (21)~ ¢ :ûb lysis *j 35ª ~ DNA cometj &V > ®î . Fig. 3öBf ? cometf j" F «~æº p~b¾ ÒFïö V tail length~ ç&Wf R2 = 0.7665 F~W ;>î (Table 2). Ò jÒ ö j 10 kGy Ò º NöBº comet~ Î· ² ¢æB head¦V tail ªÒ>Ú ^& 6N Ã&j r > ®î (Table 2, Fig. 2, 3). ç~ Ö"º OÒF Ò B J2æ~ NöB DNA comet æz¢ Jof Kwon(26) ~ f FÒ ãËî . Cerda (17)f b^º ÿ b^ö j ¦~ 'Ëj AV £ DNAê £² ¶ç> Ú ~ Î· ·~² ¾æ¾, ò~ «~î comet~ ·çê ·~& : ®b, ß® º~ ãÖº ";j ~² >æ ç Ú DNA~ ¶ç .ç>î. (27). OÒ. Ò º~ DEFT/APC ß9 jL Ò®~ b' æz¢ &V~º ¦æ»bB º ¢ Ûºf ªöº ª~ log DEFTf log APC(Table 3) 5 DEFT/APC 8j G;~& . v ò Îv ÒFï ¸jî> log DEFT/APC 8 6N Ã&~º ãËj &. (Fig. 4). r C>(DEFT)º OÒF ¾Ò ê ~ FÒ ãËj &b¾ >(APC)öBº ÒFï ¸jî> 6N 6²~&V r^ (Table 3). &Ë . ªöº~ log DEFT/APCº jÒ ò 1.08, 2.5 kGy ç Òò 2.503.63~ log8j ¾æÚî (R2 = 0.8897). Þ Ûº~ log. Table 1. TL ratio of minerals separated from dried red pepper TL ratio. Storage period (month) 0 8 1). 0. 2.5. 5. 7.5. 10. 0.0011) 0.001. 0.814 0.805. 1.190 1.089. 1.523 1.230. 1.851 1.450. 1). Integrated TL1/Integrated TL2.. R2 R2 = 0.9540 R2 = 0.8741.

(4) ®"²æ B 36 ² B 6 ^ (2004). 854. Table 2. Mean length (mm) of the comets of non-irradiated and irradiated red pepper seed by comet assay Irradiation dose (kGy). Sample Red pepper seed. 0. 2.5. 5. 7.5. 10. 91.321). 252.43. 267.22. 306.88. 315.24. R2 R2 = 0.7665. 1). Mean of 50 determinations.. Fig. 2. The graded DNA migration profiles of irradiated red pepper seed. A: non-irradiated, B: 10 kGy.. Fig. 3. Dose vs tail length (µm) of the comets from 50 nuclei of red pepper seed irradiated at different doses. Values shown are mean ("), standard deviation (bar), and standard error (boxes).. DEFT/APC 8f jÒ ò 1.17, 2.5 kGy ç Òò 2.63-4.57 º*~ log8j B ¸f R2 8(0.9528)j & . º Ûº ò~ .V b J" ªöºö j 'îb, OÒF Òö & >~ 6²& Ö " 'B . Wirtanenf Sj berg(28)º Ëòf >Ö®ö & öB 5 kGy $º ç~ Fïb ÒF r log 8 4.0-7.0ö ~ þ~ Ö". ² ¸ f 8î . ¾ Hammerton" Banos(32)~ öBº 35 kGy ÒB ËòöB 2.5-3.0~ log8j áj > ®î. ~ þ~ Ö"¢ ¾ ÝA "î . N 4B ú &Ë V*ö V log DEFT/APC~ æzº æ p~b (Fig. 4), ªöºº &Ë ê log DEFT/APC 8 ² Ã6* çj "îb¾ ÒFïö V R2º 0.9589 ;() ~ ¸f ç&&ê¢ ¾æÚî . Ûº ò~ ãÖöº .V. Fig. 4. Histogram of log DEFT/APC ratio (above: powdered red pepper, below: whole red pepper).. ö j 4Bú ê jÒ òf 10 kGy Ò òöBº log DEFT/APC 8 £* Ã&~&b¾ 2.5 kGy, 5 kGy, 7.5 kGy~ Ò òöBº 6²~º ãËj & . ç~ Ö "öB log DEFT/APC G;f Ûºf ªöºö &~, DNA comet assayº ºNö &~ N &Ë 4Búræê OÒF Ò ¦ {ö jº 1N screening O»b 'Ï &Ë ©b ¾æÒ .. º. £. Ûº~ B7 ßW(TL) ªCöB 2.5 kGy ç~ Ò òöB ªÒB Jª òº 150oC ¦"öB TL glow curve (TL1)¢ ¾æÚî, ÒFï Ã&> peak intensity& Ã.

(5) OÒF" îÃ ¾ÒB º~ ¦æ ßW. 855. Table 3. The logarithmic counts of gamma-irradiated red pepper Sample. Whole. Powder. Irradiation dose (kGy). Storage period (month). 0. 2.5. 5. 7.5. 10. 6.55 6.21 4.38 4.11. 6.53 6.19 2.48 2.52. 6.51 6.19 1.94 2.04. 6.52 6.21 1.72 1.67. 6.53 6.13 1.43 1.30. 7.68 7.41 6.15 5.53. 7.59 7.47 3.04 2.94. 7.40 7.44 2.86 2.76. 7.12 7.34 2.38 2.11. 7.41 7.35 2.04 1.78. 1). 0 4 0 4. log DEFT log APC. 0 4 0 4. log DEFT log APC. 1). Mean of triplicate.. &~& . ÒÒ(1 kGy) O»ö ~ TL ratio(TL1/TL2)º TL ªC~ Öê¢ ¸"î . DNA comet assayöB jÒ ò(N)º *;' intact cellj ¾æÚîb¾ 6îF Ò òöBº long tailj &ê cometj ¾æÚB Fï ~'b tail length& Ã&>î . b' O»b DEFT/APC 8 G;öBº ÒFïö V¢ ªöºf ÛºöB jv ' ¸f ç&j & . ç~ Ö"öB DNA comet assayf DEFT/APC O»f OÒF Ò º~ screening O»bB, TLf {O»bB 'Ï&ËW {>î .. 6Ò~ º "VF¦~ ö¶KBBÒë~ ¢~b > ¯>îb, j æöö 6Òãî .. ^. ò. 1. WHO. Wholesomeness of Irradiated Food. FAO/IAEA/WHO Expert Committee, Geneva, Switzerland (1981) 2. Codex Alimentarius Commission. 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(6) 856. ®"²æ B 36 ² B 6 ^ (2004). of the European Communities, Luxembourg, Luxembourg (1993) 29. Hammerton KM, Banos C. Detection of irradiated spices with a microbiological method DEFT/APC method. pp. 392-396. In: Detection Methods for Irradiated Foods: Current Status. McMur-. ray CH, Stewart EM, Gray R, Pearce J (eds). The Royal Society of Chemistry, Cambridge, UK (1996) (2004j 7ú 27¢ %>; 2004j 8ú 26¢ j).

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