___·_ i· · II Ii i! Aw 131q ~1 I ·'1E ~II ~·!f.! '· · '' ,· · I, 1 ~:rfji hi i··! "' :I! ~liil~ I' k:t :;I~;·~paarmranuu~a~9~ I el I la~Il~BltEfi;~ Ei!i~b~M~e~a~lC~e~e ~s - I ~c~knwr~l~ ~:iil I : r ,.,., ,,,I~ :1:Ilit .i, i:liIl II ·: ,,,, "I ~ · ·~ ~''~·~· ·('·'I '··ll:I · ~PIC~ -~~a~iT?~ :::' I i.; 5~#dLtBISlgPU~e~l~lY·"nsm I -- ~n~sPIYa~PF~ :1 ·I I,, :: ·t~lf rl~i ; I tl ,,,4 1 .,:%?l~:~J~s~W~IJ1~BF~e b~L-, IRY~-U~Ui~ r Y( II ' 11 'I I ~t;f 1 I · ,, a i: O rU (rl \O a o IE~ Irl Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 YI RESEARCH AND DEVELOPMENT DIVISION MONTREAI THIS REPORT MUSt NOT BE COPIED DR SHOWN TO UNIUTHOAOED PERSONS. O C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ;dle *' usoru~ro~e REPORT NO,_lP2, cow NO.~ IMPERIAL TOBACCO I~~ RESEARCH a DEVEmPMEW DIVISION MOKIIIEAI RCrrmC1~ED EmYMAne DEPROTEMIZAnON OF VIRGINIA TOBACCO Author: M,H, BIWMORIA IsJurd bl: Dr, S.R Martsy Dm Lwed: Augw 13, 1993 DISIBIBOTION: Dr. PJ, Dunn Copy No. Dr. S,R Mastey 2 Mr. G,A. Red 3 Dr. R, R, Bs~cr 4 Mr. T, Riehl 1, 6 Mr. T,I, Wilson 7, 8 Mr. W, Zapf 9 Dr. E, I~uJeh 10 Dr. CJ~P, de Si~ucin II libruy 12 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 WBORATORY REPORT NO.~e2 COW NO., IMPEBIALMBACCO LTD, RESEARCH a DEVELOPMENT DMSION MONTRUL RESIRICI~D aaYMAnC DEPROTE~NMTION OF YIRGPJU TOBACCO Autbot: M.H. BIWMORIA kR~d By: Dr, S,R.'Ms~y, Dps ~wed: AukJn 13, 1993 t W Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 SUMMARY 'Ibcplaraaof pmDdm iP lob~eeo i mmidend PPdcsinblst only bca~ tboy a pnaunon of cain no~c, in ~o beatw of their L~iodPian with poor ~liB smolrr, 'Ibb mpoR ~muhcr at Ofm eAbrp to depmldire fioeaned mbum mis mmmacilly miieble emyme prcpfit~n~. at sPdin mated rhov thnl 1 vPiey of miaaba prcu~ a nitlble for motel mmonl from fieenued mbrm. paiatim memjon rhmJd he mde of e Sigrm pPatrp~e obPined from AIDcmillm o~ne 9~ cfficimly nwwcJ Ilp to ?OI of the totel aiaogw lad np to S3n of the bb~ceo protein within ~ boom of b~C~C~t. RtdURiW( in mOElgePiCiPj Of SmO~C ewd4wte of up to 10x k 1~ beeP momded by both h2nrrill end fuP~ emymr pmmttapm. L apcn3ive i~dumi~l enzyme p~epultioar supplied by NOYO hrJe ~o been found rmitlble for taduaion of tob~co prtcin, P]mprm~es luch u Fiein, md 1Sip pm(orsobaiP~d from ppyq wn without rtiviry under our #rpcrimeo~l cwditiom, FUJ, mb b belived to be the fan ~a~dy ofia ~ad in which I ~Yect d#erminltion of protein (proerdute devdopld um) v~ used to monitor prMdp rcmwll from tobaeoo. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·2· gMWORDS Virgioi~Tob~ceo MahPol·Edneted Tob~eeo Sdi&Sttte Ertrrtiw DepmtdPiz~tiw RotdnDaetminPian Protsucs Eargm# Qgarcoe Smo~e CWdGPlp~ ~mcs Mutageaic$y 1 Crl Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·3· ~obven~I ~~~ilynmiill~l~~~iel e~~~ ~ ~ C L nudn I ~ ~1~ ·IPI~ r ~ ~ ~ codaPr H~ Carp~,~~ ~ ~ ~~ ~~C r ~ ~ r ~ ~ ~ ~~'C ~ C ~ ~ ~ ~ ~~ y ~' · ~ I I~ t p0dnY4 ~1* ~ I I I ~ ~~ i I I ~oldi~ ~ ~ budhtil~ ~I ~~ iC ~ ~~ 01 ~ t i u ~! r ~ I~ L~L ~ PZr Y C ~rin~ ~ ~ ms mmikt~l Ilachbtiol~~ 4'Y1 ~' ~ pC6ru. f k ClibPDF - v~~fastio.soni MATERIAIS I MFIHI TPblPPP ~ f~bC~d lo~Lrm, ~ ~ obai~d L Ihiddid,C ~ I~ ~ fobrcd L 198 ~ 19n, n ~ i ~ ~ia. TrmFj·mne mrjus, tardad L ~ IC ~~, I ~cna~d t Y 1S~ ~I~ ~ L I~ labrm. ~e sowtn of ~b 1· e~ewive protrpcs ~ ~ u~~. Amonl ~ ~ ~mbJre L Emyw D~vdogma ~, I.I, ~lpirpra;cl ~ ~ ~barlDsic~, ~, ~ ~ 4~ ~~ TrrPn~ Ttc~ent ~ Iwied ~ t RIIPCI~ ~ ~ lr powdad tob~c~o fl vol~rmc~ ~ 3 a Y @LwFh~tc, ~ 1.); d~te, P 1.~, ddin~ etelrrm ~ s~io~ r stitig II ~ t ~ sui~blt tePpen~Pre Lr periods T,~ ~. l~d~ s~diLI C p~atiti# ~ bb~cm) ~ cmied ~ e eonicll lib, ~~ ~ scllcup ~ilog~ pPPtitie~ irvolved ~ ~t locllly~ode ~· ~ crmrtion qnrm C Filure lr ~ ePzgae t~e~tmcot r wried ~ t ~Z ~iseue, Lltobrco iJ ~ epzym~ ~ plwd L ~~s ~ ~ iPeub~ted I n tcmpa~ure. ~~ ~yne tmtme~g LI Db~cm ~ w~JL~d Y ~ J·la ~ ~ ~ Y~ J ~ i ~ Ya or ~f 4. Sp~dfic 1~ I provid~ ~ LI ~~ re~ults, Clraratts~ d~m~l apnaeJ ~e ~d ·4 1 Idly·aud~ di sllbble br ~ ~ ~I ~an~ d ~~, YI eoadw~Elses ~ prepwd s4 LI H~d BgwllL ~la )~ rmebie ~ lr9~ t mmyaie tntint ~ lesaibed d~ gS. 1 N ClibPDF - v~~fastio.soni ·5· 17brmicsl de~am;nstloa~ Twd nmpa ~a~ dmmiwd by tbr I(jdY mmod ~da~ Iho gjdlce SF I lp~ ~dlo~ ~a m~od pmvidsd(l6]. Radp niaogra aro obPiatd 18era~e~n~it~ OJR ic d og'C k tep mdnw, Rotdn nm dasmlacd by n r#c~t~e~dopsd dittaproc~dn~ (1~. 'Ibe ndmetoom in Irtll ~o~en lad padn me aprmKd g Pm eem dIl add~o Id mt on ~e bais of the o~i~in~l mi~ oflob~ceo aaotad Muranmie SbJdie~ Moppicirj of Be smo~e mndmstta wot ~anmina~ by ~e ~md~d proc~dure follnva in u ~bonay (IS) usiP~ Sllmwdll twhim~nium T~I, hbmy pmv~sd by Rofetcl B,N, Amn (II, 19), StPbdcJ 1P~gds of rbe m~agmiciry dan Kls cPritd wt y doc~b~d clrlitt (15), I \O Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 RESUITS I ·~IISSld Sn#oin! ~~~ Pmteun Ib C Im 1~.2 ~ d~la ~ Ir ~n~a C r~ mnd, a r pmcaxs ~ ~r MildlL CowCentlL t ~ IY ~YI I ~ I Illba ~lpmt~eJ IY L LI ly dCL L ~ dbtind ~ Le h ~e~aeainl ~ rcc~d~d C ~I 1 h Y ~C t ~I ~~ ~ ~ Irl ~ prepPP~w b ~ ~ II 1 ~I ii 1~111 ~II ~ iaqm ~ ~ova ~ Y ~ ~ ~ I I ~ ~~~. ~ rmtrq ct S~ni~e~ pmt~w,b ~ ~II ~I~ L b L~,~ ~ rewwtd ~It ~ 81 ~~ It I ~ p~ ~ ~~ y~ d~~ g~~ ~ Ln protcur ~ m~itlb~ L~ 4~e ~~ · ~~ ~ e ~ia a n, ~ ~~ ~ ~ Ltd ~ ~ L~ caz~a r ~ L rewvti ,I~ ~ tob~ceo. A ~ B.~.t, Snth~mpo~ d~ LI a~ asentat IYr dr ii rerwnipl II grmafi~l pmt~cr 0~, ~ 1~ YaLIY aP~doa Ik ~ ~ uam 1 td~cfo p ~ poea~ C ~r YI, t r Idl~ r ~ ~1L· plmcucl ~a ~id·me ·ditiou ~I to~cco ly, h~ LI de ~LL ~h prs~d C ~qll~ r ~ ~ i ~~ Irolcua ~hbwrd 1~~ d ~ ~ ~ ~~ LI, ~L ~ 1 ~ pmteus 11~ L ~~ ~~· e~rl~w a re to~feo ~ ~ L ~~ m ~ Ircscrr d YLlpl ~ LI ~a. Is StPPPPYEe~ ~ dlPLDIIII c~tyPa ·, pnfo~sd ~, ~I(I I ~~ ~ ~ C r ct Jld. I SP1PtPIIYE~ pnpuri· ~dd~ Y ~a ~I ~I le#Nm ~ ~ aptiul pa of laiv5y ~ C 11 ClibPDF - v~~fastio.soni __ Effect of Olarhine9Red~6rmpf Nihwenand ~uPeenieirv h a olimmt 0 daamine qPMimively $ diflPCDfe made by vP~iag of mr~bm~d fobaceo uoda w apaimrm3 eoo~i~iaoJ. ~ir ir recMdd in We 3 ~vb#e it d be lecP thet luam c~2~mMc~od IObeccD a tVbea, I ~ilaifjaal rddpet~n ip U tool olOoea md Amcs rrmt~gem'cirl sre noted, w~erw le unmrhsd tobacco bowed no 8ucb reduction, ~wl·Exmaad Tobacco andPrP~ea~r Aaivih TBe MIlPO of aaymMaod Ic~cco muh~ i nmnrl of aieotiae lad nan. crm,[daed dninble to prezmsd ~ob~ew wing mabaool whieb nwves con~idenble Imcow of mbacm Piwtine d rukn. ~r mnbeool aneet muld lba be EonfmPltrd Ipd edded 1l to me Vgbsd emymMeped tob~em, Ibe Fstion Beo wu, wdu]d 8ir mc~wla~c~ mbrco be a sahble rubsr~ee t tbwe Si~s pmtcaa? ~ee Ig~in, 1 comber of protrara wae ~a~ePd for PmPiP I0klbiiblti0l IDd me nsults ObtZiKd oe praent~d i T~b~ ~, Fmrm Lr~blc, twill be SkP thnt dr tbe pm(eeea tcacd wae wUc for removal of gmrdn from mc~wlznaaa~ tobacco, Toblleeo.Rnrin Spiubil~bt~OPrnt vasw PnvdardTobarca fbos fn, 1II our ~io vac Pnda~ea wlPe an mbPcco ice mi Iwm i plillbk fD[ cipr~t~mJdo~. Hnnva, it a Mt ~ uK of poa~dcnd mb~cd by vpll of pmrmiPI ~ ]Ppa ~ufice a~ wol nnl h I mcse Idrm nmonl of si ~i~y huoh~bll lobrco pmtdP. ~, vba ~l~dercd mb~cco a wd, Imd Sigma rm~ma ezn pro~dn rcmwPlr gnaa tbun WZ a ~gYp LT~cS. Tvo almar, ebamiel pmt~ mpe ~V) ad IproP1K6nm la~dUy~ m. pvc 1UCI Of 711 1Dd 711, le LiphcD lac htw ev4 raorded ip our aryme smdia. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 Serspinn~~~nt#Eo Ilyl~ ~ Rcovd dlval c of iapaok pmtr a YI~, ~1 ~II ~hLara Yf aPlIld ~o vail a ~ ~ far ~ ~ ~~ E~aa~Lc, pmid~d by ~I Dmlqma Cdlp, N,y, y,ql ~I i ~ liopl pr ~· y ~)~ Ep~obJ;b tobaeco t Ilb, At II mi ~1~ ec~ypelpn lobaao, oPIy lyt ieram i niWIee remvll oeaurcd nltive ro I, I ad farkll unma 1~ wed ~I apd ZlI aioIen rcmov~ ta ~~~ h pepai~ slppli~ by L Lbwrwia, IPc, Ime ~ I" a ~cord~Q in Ye 1 From Lr ~, t~ d· ~ LI I· apswiw #Ilmmciil Iwre~ - a are ruiDbIe ~ Ie agS In ud npeaslve 0~ rouna md IriIsaeI ·da ~Le ~~ of eoPIIciaI ~oY·. ~daemaar ~acr#ise~~C~ PmtdnR#nwal hr ~d rtempt ~II ~lr i PCKICC Dfl da~fent, sodip Yd ~~, Y Iq· i larirl7 employed I mla proleim fom I~dl fnmor obtliaed hi d ~opo~ato, w laopn man)e ~fll md SOI Inc~ld LI Sft~PIIYEPforw ITyle fW) d tbe PI ftem ~ls Lemnmtml~~ mpe X), mep, C y ~ J ~ ~ for Ill la i II ~LL IcUlpt b Ij~o~cl ~g a Iceoppmi~ II ~I i Ames ~ of7li md II t g XIV ed I nspcoivdy, ~aatiylX It data Yltd a ~i~o~a maVI ~~I ~1 ~~ i ~ ~ IljZ~ i Ir mapu I bi pmtei ~ ~ ~II ~bLtio~u oI amer, y IC aod Novo, 4 d~, rd Le ~I dd i Ye ~ R Se Tebl( Il will La h i Ir II ea 3~ djPriOW oI Imtc~e~ pve r i~a iPncws ovm ~Yr ~ b ioti P(PMi0u in aI o5 lowevn, wlcl U~ ~ Mg I c~Elplol~d rP t ZS mIlIm tob(ceo, a reductioa oISII wat ~d, ab r · I$mvawt m the ClibPDF - v~~fastio.soni __~____~_·_ _C_ 311 ud II d~ ~ b yl d Nahw, lnp, I1, dmr Eolecpada I rmblfao ~I of L mn alcuivL kt pnr SiOm a~yla, i, hP~e pmce~K A 9 rd I pmtwe ~ I BLE1191 1· mPe Vm), tao, did ~ lIw mi impnn~emea ~ II ~~ ~I Y Kp*rd~ I pmd Raonl pf PI a ~~ t rL pdi~o o ePrpnd to60I ~ 711 rcewdrd ~~~cprPd~ CY, ~. P~nrur ?trlrmn ~To~rc~Scll~ Y Ie ~lr sCdd ~ L a Emitd aU r pr Id ~~ ~tob~cm. WiI Is r ~I ~ Dor ~· rid ~aba IF r ~ Ic epILitl I raIe ~1 lp Yp ~~ ~lob~te~. In Ir ~ c~aiment 14 u ~ ~r 1~ la ~1~ ~~d·and mb~cm ~ II~ olvrta % he~YP~ Pm(a (S ~ lobrm). 1LC qi~ d~ ~ YIL vI obti~d ~ Bl~a~naio~ i tnPI abP~ YI La ~tmtwt L parh ~ "c(d y 0 LI ~ b aa nciY dc~e~a~d poadrr P~ llmpof30I ~1 'nmtad~]~~e~uae~cr hr~~tob~o~~~yl~~ ~~~· Poil ~ ~~· ~ ~g I~nia 9~ rnylc d~mnt~ t r pmLPLly ct I ti ~i~ ~ lodpcsl b Is ~ yL I~ t tk ~ dI" famcpa. 91 nyre ~ ~~ ~,~ L cilmns vLieL va Ia Y fm L nnlelq, k y(l II~ a ~ I BZ nLx6ol~ n~tllmic ~~ ~ " nsorded Y IL of pouw rm~q ul IL Il 41 ~ ~ s IOlU(( PrmPC btlllll Q · ~ i ~ IOVbill d IIPrCdC Y, Y ~pdna d~ lawad lh I d~ , ii iud ~~ ic ~I lki4 L ~ raard Idotray, lad um ~ ~ ~~ i~n~p~ove Ii lroa, Y i~povana r dclIy laannnbd i II la apduq i rauIII Df vLIm I newdsd i rcL Al Ii ~, I, ~·~ I y r ~~~ y~, ~d ~~~~~lh~. ~1~~Llmtululpmmi~r,~ e i ~~ ala~ I P'S ~ ~i~i~d LI~ ~~ d. ~ I , ClibPDF - v~~fastio.soni ·10· In Y #cond dwp clperimad we reoorded I nX ndudirm in atl ni~o~m ad 42 raduetionof pmtdo cwtePt in tbo tobrco laSds in 2 boom of t~e~nmt, Trc~eat Pr I f~J~e~ 2 ~opn led to In~mmcn of 691 aiaosm ad S3li nboco~on in pfitdo comcm, Scb k e dlainn implovowa over dr hm enpait ram PO fidml rednotion in nib·ogm tva neordrd in I boar of t~c~e~a FWIKI, this ~rd~P Of nipogm otlbad 7011 cDmp~ hvoPmb~ wah hn~l~e~ion~ Obtlined ~ui~ thh ~P~t~l~ fatar ~M2) in d~jcJe wrOiaa, Tob~eca atnet~ umplrd ~wn this re~mon mimue a rep~r iman~ no to Ibdnn IboPnd r ~dwlly iaosainr niaogea rmnem w6icb raebad a in~Pcuc of 451C ova eon~ol in 2 ~o~nr ad ovn 702 in I hoarr ofenEgme m~ent 'Ibr aayaerrmed fhraurd tobacco wa mde imo dgmaLd, aha WhCP tCltd fm Ama muhgmidty, sbowed I1OIC rrdunion in spaific letivt9 a 2 ~6un alprcreur acam~cla. Ilowswr, funbrr Imaoepl did pot nrplt in f~s lomring of mrm~idrj. In moelp~iom, than edits ~ow tha I Maiag of microbY pmtrus Pr suinbla $ pmteip naval i ~ubanslmbrco. Pa6cniaiy rtive is n Stleotomvcn ptrpz~n (Sitmq TypeX~ but in price Cwloda in a sMlon M li~orpaj de, Ye 1 pnparmbn abaiosd ~cm dspgpillp~ ra~e (s~i~ Tgpe ~ is eito hi~~ ativO on lob~em bnt is in~xp~mk arrmgh to be nad in I~borPay de snldia. Hnvmr, if eva mi~ pPOcsdme is doptb~ On a iod~hiPI rcdc, thm Nwo prep~t~nr sPcb a AlahK end SniPuc will ~Ne to be Iwd, hurha, plan pnftcue~ aor6 a Fidn, ad ISigmm pPepwioP a~tri~ed i PIPly4 war twlly nnruittd for ptozrim ~o~ ~am BPbamd tpbwn uo~ no ~aimcn~l madit~nr. Fhuny, Iltbnyh # IOtl] ~j~O~rP Ind prm~ d~miPln~m ue D~tt~blC indiemms of the cffidmey of pmtws in proeiP remwl L ilnooaed 'obrco, ~ Ipla procshun, on lixarm d r ~edhhl, Mould be Le m~od of cboip, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 -11· ZIEF~I~ 1· T, C, 319 p~l, Bcia, T~b~fmJcb., 1 190, 1973. 2. 31. Does Bi~l j~LD~. 3931991, 1 21, 1978, 3, D. W. De Iomp epd I. J, km, rob. Rcr,, Z 1, 1979, S, Miplaald gll, Mutlt Res,, ~, 319, 1027, i, T. Mmmaoaa ~b~ Ihje 36 281, 1978, 6, S, Stto ga, 3311181, RCI., 6(1 1S5, 1979, 1, D, Yo~id~ nod T. h~ma~o, CPnea Lat,lQ, 1~1, 1980. 8. T. C. Ib gal, Bdtr.5lb~oncb,,~ I(, 1975, 9· Irlre~ Ibid,, 11 63, 19n, 10, t C, T~o, Pmduetiw, p~pio~ogy red Liorhasi~of acmbsem pina. Idrp~, lac., Barnillc, MD,, 1990, Il. T. G, ~hell red O, L lobmon, B.·A.T. SoPt~mpran, t~p~t Na. RD·IIW, U·5·19n. 12. D, b ~ntin rod C. E BerwtC 8.dT. SO9tbllPplo4 Report No, RD21JJ, lf·16191. 13, C, F. Ilentl, 8.·A.T. SoP~mptdn, Report No, RP1210, ]M·1992. II C, F, Hnea md G.Ringp~iPg~a, 8,~L~T, Soum~mpmo,l~pan No. T·235, 101·1992. . Is, hi:. a, ail~ori~ In~eri~l Tobrem ltd., Rame~ Report No, 162, 13·5·1981. 16' I~Je~ procedun ~i~ Kjdt# Sgstrm 1, T#smr rmmul, 19·9-1979, l't. M, H, Blimori~Papapacms~ st ~e 161b Tobwm oemig' ResMb CPntPmee, sept. n· 30, 1902, Abatnet No, 21. 18. a, N. ~ma gd, Mum Rm,, a 317, 1915, 19. n. id. Maon Pd 8. N, Amre, mjp, 113, m, 1983. C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·12· Idlltt ~hdgPL~IIIP~I~LphaEl Irdl~Ul(~iLl~tsl~t~L~IPIPiE~: ~ rw~. h~rrlnlr~ ~ 1 u~ ~t~nD~t ~LPi~u·B~ I~ a ~si~u·~~ J~ a 'gentrrmu·~n I~ J1 k"le~p~n~ehtieul·~X I~ a~ a I ~t~LI·b~ I~ hrril·~a I~ O LPP~mrm·ba I~ I~ n ~n~P~arm·bn a t~i~·tia J~ # Srrmi~ s· · ~w Ir R g~ ~L~L~·~W I~ Q I~ ~~·bw s~ 11 d~n~·bl d~ei~F·b~ a kt~i~i·~~ I~ R a~ n U~IPe~u·ti~ 1~ J~ 1 II L~~"cYlr~,,,,,~~, e kl~llllLC1~~1Ly~y ClibPDF - v~~fastio.soni ·13· TABI~ 2 &e~hdiandtohcpeffpkia tad oldrrmntusidh b~ wakuc ba~af d~ae~aPsb~o~~eaelmder wild atc~aoditioP~ll(~kplai~tlpc~ ParrPt r6dudioo of Eaz~w 29tal 1609899 Mulynidtl Bacldorlichmifwmis 39 99 T)pe W1 StreomwFL~~iw~. 21 33 Typo XXI ~soc~lPra~c· 29 63 Type fWII Fido·M266 12 Nit FiciP·E6038 N1 Nit P~yl·T~pcm K1 Cut ~ut·currd tobacco ~YZ~ b~Qt~ ~it~ proeew d 5 m~ mbaceo Ilypr XXI 3.1 nylgm) rmd 1Z m0isDlre cen#pt obPiwd usio~ distilled wster or p~osphne kdfa, plt 2,0 (Type III lad 960081, bI the md of 18 ~oPn incPbaioo the toburos were ~kz~hrd twi# with bot'AWar snd dried, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·11· ~DLanfr~b~ lr ~csmd kkrm~k 'I ~~ t~beePrkkemni~ .1 ~olte.n~utuaieiC ~eent re~Pdl~ ~ T~hat ~E~LP~S rOblCCD IllfbCd 21 4 ~ La m fobrea r~ 1~ ~ fEawd tob~eco ~ trtxad ~ SL~B11 POI#1C I I ~ IOblEeD ~ ~Z I~ a~aio~d ~ plo~plte Y ~ 2L r ~ ~ ~ P b ~ PblDW ~I tMI~L~d ~ r ClibPDF - v~~fastio.soni ·15· TbllLE I S~ninr alSimFn RO(CLICI for TOblPEeffOtCil Rgnonl ud Rcduetiw.olSmPI~ Mu~idtr tim ~nabu~o~hrdedla~lem Epllme rwr# Paceat ndurriw of O ~oaaatntioa· mglga tob~em To~ Niogro Mutbeakitl S~mti~a~s · Type XIV Img 11 60 B, thwKIDm~eo~vtiolt· Type X Img 48 Img 58 68 Boderiol · Type XW 5mg 61 n E~aeti~·Typc;lWV Imll 49 5mg 59 67 B, wlvmvxl · Type XV 5mg 35 41 25mg 45 Ii. li~heoifd~i~ · Type WI Smg 35 44 gb~PPO1~ ~~ · Type WI 5ms 28 25mg 53 h~ao~ · Type XXm 5mg 30 lo mg 26 48 ~b~pjp~ · Type Wt 25m~ 33 43 MdblDo1 CXt~C1~ Aucaysd tobacco wet s~peodd ip 20 volumes pho~pb~te or citntcbuff~ rod b~WLCd with the e~yme with ~ing st rmbimt or n"C for IZ·18 hours. At the epd of the trc~oaa, the tob~cos were ~P~hed twice wah hot weer tpd dried. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·16· TAIIIE~ LPyjlll~l~nlad ~nrsLuhea ~ a~armti~rrtri~ maPnl ~t nhrllu ~ Y ~~ F~tlm~ ~Wlrm ~nknl TP~ea Ypac~ 81 91 dmra4usn~i~ II B~QIYII· PJ wJ StOppp~E~Pj~BU ~I QI Bdlntli~E~r~i~n~pir rJ sJ B~E~bYb~p~n~ptpplaip~ I) II ti I B~e~ai~ 61~ ~ Iwv ~ r ~~ tob~aa ~ ~pcad~ i ~ Mhurs phosphrts ~ ~ t)l ~ ~ r 5 ~ tob~cm ~ ~ 5 mb~em cPzlme prepmtioo r dJrhl t It ~ t ~ Jn~an ~ Ic ~ ~~ ~ ~ ~ I ~ ~1~ r ~ ClibPDF - v~~fastio.soni ·i?· Tdl~b Sctrmiol o~ NPm#dafos Ipr lo~eeo watrin rana~slmd amdto rrmtua~idblad~djpp PmJrm oaI roaotatratIoo · ParrPt raIud/oa ol mdon lob~eFo todPllmpP Alalw 5 12 50 48 100 IS 200 n Nnmsse 5 33 50 11 100 48 200 49 SniaPte 5 11 50 51 loo 55 UWI SI Ronrt eypsin 5 1 M M 100 a uYI 47 Ca tobacco ~ rplpeadtd ip UI wl~me~ of ph~aph~te ~dIe~L, pA 1.0 nnd arusd 1 emymr ait~ Ih~ng fa IllhoPn psmbimltemparmre. At tht rad of Ihe ~e~rmml the ml#rm w~ wnsbad rwier ~ith hot nPa ~d Ihrp drid. Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 ·18· TABLE I Rcdndioa p~lob~eaa wddn lad ~6~e mut~renld~ hr~a~nt with dinarat.prot~se ~ombi~ut~om Edllw aod ODO~Ob~aO · Beecoat rtductiaa of mdPa tohaeeo total Pitro~n Al~sc, 25 mg 35 NmttMa, 25 ny 43 Saviaarc, 15 n~ A Alduc, 25 me + Nr~w,lr mg st Alc~w, 15 mp ~ SwiPuc, 25 mE 48 N~A~SC, 25 mg + SlviPz~c, 25 a~ 51 b, on~, Type ]a$ Smg 60 Illiehcoifwmi~. Type Ym,5 nd 70 Spe xlan, s mg + Type Vm, 5 mg 72 Cm tob~c~o wm suspeoded iD 20 VoIPmms of pbWphltd buffet, pH ?.O aad aePad with ea2yms(s) with ~h~iPg for is houn at amhieottemperatere. At the epd of the trePmePI the tobacco wm wished tsli# wi~ hot wPa d thep dried, C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111 ~ ·19· Td;Bl~d Rdrin mranl LI anw tr#t~neP~ad ~udio~ ~b ~uturnidh· ~M, 1 ~IZPME mf~L NITIIOGhl IILOTPM M~AGEMCFIY . TrtPITMPNT (MGICM) S~PBQErIe ACI1)TI~P TMe ~) ~I ~o IA a~ oJ 111~ ~1 g) LQm LO Illaq aJ a 14r ~ 10 a3 ~ L39~ 11 411~ a~t L7r ~ ·1~ GIVEN I BRA~I(E~S I PERCE~ REDUCI1~ ~~ eOHIIIO~S ~GENIC ICmmES Re II DIFFEREKT IUWISIRW I SIGNIFICAKIZY DIFFEWTT ~ ( I$ ii ClibPDF - v~~fastio.soni ~CI_I_ ·3· TAIILE r Pmtcin all ka~ h·J ·1 drrtion J ka ~alaid~· bkal MZYh~ ~L MCIIOCI ~OTW TREATMEN~ (9 ~GIGM) SIIELPle ACITVIIY IIME ~ Co~aol 1W 43 QQL el ~3 0) If ~ ~O w.o ~ lo LII 04 3rl ~) LJ ~ at or g) 2~3 6) P1~ b9 Qcc~ II 1~ n.l O,W ~ ~MBERS GIVEN 1 I~Q[ETS ~ PFfL(WT~ REDU~O FROM eO~ROIS MUTII(ICmEli FOUxlWn,~l ~ DIFFERWT SUPERSCI~PT ~ fB~CANTLY DIFFERENT ~ ( (~g ClibPDF - v~~fastio.soni I~IG(IR~ ~~ ~ rxr~~ ~w Rr ~ I grir~; ~8~ VLIYI o N r'B Qruulrs7uu a o ClibPDF - v~~fastio.soni i. ·~ · · I : :i r·*.: ·r;:·':·:' *'· i ' 'Ae~' ·· :;:· ·: ' O N C U1 Clit; PDF -!::!!::!!::!.f3 StlC.i: 0111 " 'L·' Iri I ' ···~ ; I· 1. ;; 1. I iii :· 1: ·1. ~··I~ i:i 1:ikllf ·\~· ··i"r 'il' ·I·e:;i a·; :,I ·;·s;i : 1 ii 11,~\5C(L' ~: i;EUElf Si; 1 ,:~:Ei~irr~~ '·:v;.r~~~L~T~,h;#i(aRHE~~~M~IP~YB#B~L9WF~U?IIP ·' 1 ~:'fiS: L':a ;1; !·1 :!::J .·· I ~ ··;r~ ~I '' " wi!· ~~ · rll· I· ~ ·i ·cl I. ;'l~;)~t'fcil~E~Bda~~'"~C~F"'~·~' ~nY~""~BRERA~·~MAF4TU1B~Ftl~ti: : ''' ; ·''L ;i ~~ ·1· ~(:1 UII1~~OII~~L~C~I~ ~JISI~IG~·~L~PI~·~~21 ?IslYIYTII~1Y~Y~ !1111*r·lln·1~ 1~. 'I :I ::: O IV Q O U1 \O C: I i I; PDF -!::!!::!!::!.f3 StlC.i: 0111