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Nex=, ~e ex=a=ded =heso s=udLes =o include burley, dark, ai=-cursd and
dark, flre-cured c=bac==s. The cgsul~s obtained ac 5 ~ enzyme/~-m tobacco
are presented in TaDle 2. F=om ~his table £t will be seen Uhau =hls
concentration of enzT=.e releases Lit=Is or no protein from the hurley and
the da=k ua~ac=os. On :he other hand, flue-cured tocac=o showed protein
removal of ac=uu 50% u~der the condi=ions of the experiment.
TABLE 3
ENZYMATIC SOLU3IL[ZAT~ON OP PROTEI~ ZN D%FF~R~~rT TOBACCO TY~
, , , ,,
TOBAC'.O TYPE AND PROTEIN CONTENTT -
HG GE~ DWB
TREATMENT T~ME - HOURS
CONTROL
"NZy,~LE-TRZATED
FL~-~-CUR5~
2
4
6
2
4
6
DARK. .---_UE-C~D
2
4
6
2
4
6
85.0
99.5
89.8
158.0
165.3
167.0
231.3
227.3
234.0
210.5
204.0
192.8
46.8 (45)
45.8 (54)
44.0 (51)
162.S
154.8 (6)
151.8 (3)
229.0
221.5 (3)
229.3 (2)
190.8 (9)
201,0 (1)
IS4,3 (4)
t IgG as s~andard.
The =obaccos were ~=sa=ed with A. orvzae p=o~ease (Sig=a Type XX~:I)
~m'a to~ac¢~) f:~ up Uo ~ hou:s in an environmental shaker at SOeC.
The numbers in b~acko~e are percen= reduc=ions.
(5 mg
B.A.T. CONFIDENTIAL - CATEGORY I: ~|INNESOTA TOBACCO L[TIGATION
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When the ~unga! ~r=~ease :cncsn~rauion was increased to i0 ~g and 20 mg/gm
~obacco, up uo 7C% of ~hs ~r==sin in =he flue-cured tobacco was
solubilized. Zn contrast, only insignlficanu amounts of protein were
solubillzed from the dark, air-cured and dark, flre-cured tobaccos. A
liB:Is more success was c==ai~ed with hurley ~obacco, with about 20% of
~he pro:sin being ~emoved a= 20 mg pr=uease/gm tobacco. These results are
presented in Table 4.
TA~L~ 4
ENZYMATIC SOLUBILZZkT~ON OF PROTEIN ~ DIFFERENT TOBACCO TYPE~
9ROTEZ~ CONTENT -
MG/GM
DWBt
TOBACCO TYPE &
TREATMENT TIME
(HOURS)
~UE-C~R~D
2
4
6
DARK. AIR-CUB~p
2
4
6
DARK, FTRE-CURED
2
4
6
2
4
6
CC:;TROL
IgG as suandard.
T~.O
7£.25
74.00
147.5
13~.5
1;~.0
194.5
i$~.75
166.25
165.0
15~.S
*ENZYME TREAT,~LENT AT
I0 MG G~~ 20 MG G~*
25.S (66)
25.75 (66)
24.0 (68)
131.5 (7)
134.25 (5)
133.5 (S)
196.0
193.0
185.0
159.75 (4)
145.5 (12)
25.0 (67)
23.25 (70)
20.5 (72)
126.75 (i0)
130.0 (8)
127.5 (10)
184.0 (3)
185.5 (5)
184.0 (3}
139.0 (16)
134.5 (18)
141.25 (11) 129.25 (19)
-Prouease (Sigma Type XX;;Z) t:satmsn= a¢ I0 mg & 20 mg/gm ~obacco for up
~o 6 hours in an environmental shaker at 40"C.
The numbers in brackets are per cen~ rsduc¢ions.
~. Hvdrooen Peroxide Treatment of T~bacc¢ U~der Solid-State Condi~icn~:
In earlism s~udies we showed cha~ reduc:ione in Brotein up to 20% were
obtained when cut, flus-cu=ed tobacco (CLX-87) was ~reaued winh 3-6 ~unole
H:O: per gm tobacco a~ 50% ¢oisuure for !S hours au room ~emperature.
La~er, metal ions such as c=p~s~ and ir=n were added a¢ a concen¢=ation of
IO ~mole metal par gm tobacco. However, a~ these concentrations of ms~al
ions rapid heating and char:in~ of the f!ue-ou=ed tobacco occurred, with
copper ~ringing abcu~ a fasts: reaction than iron.
j~
P~
J~
P~
B.A.T. CONFIDENTIAL - CATEGOP~Y I: MINNESOTA TOBACCO LITIGATION
BATCo document for PFSFC 1 March 1999
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Recen~l~, we carried out these reactions a~ lows= concentrations of iron
and c=~per and were successful in =smovLn~ up no 40~ of ~he prone£n from
flue-cured tobacco (see Table 5). We ace ~:w ~epea~ing chase experiments
an ~C~ and 70% ~ois~ure levels. These studies may also prove valuable in
~mpccving the appearance and keeping ~:ali:ies of poe= quality nobacco.
TABLE
EFFECT OF OIIDR.TIVE TREA~'~ENT ON ~ROTEIN CONTENT OF
VTRGINIA TOBACCO UNDER SOLID-STATE CONDITIONS
WEZGHT OF
TREA .-.~.E~NT
WATER - CONTROL
H:O:
Cu: -
Cu: - H.~%.
Fe-'* (i ~mole)
Fe"~ - H~O:
¥e:+ (.= creoles)
Fe-~' - H:O:
TOBACCO RES IDUE
36.0
34.0
36.5
37.5
39.5
37.0
41.0
34.5
P.~OTEIN CONTENT
xsl~x
TOBAC~3 RESIDUE
TOTAL - MG
93.3
63.3 (32)
86.2 (8)
55.2 (40)
81.3 (13)
59.3 (36}
81.2 (13)
67.3 (28)
3,359
2,16S (35)
3,146 (6)
2,108 (37}
3,200 (5)
2,213 {34)
3,329
2,312 (31)
Cu~, flue-cured nobacco was treated w£th H~ or H~ (I0 mmole/gm ~ob.) or
Cu:* (I ~mole/gm sob.) or Fe:" (i or 5 ~mole/~-m no~) or Cu:" - H:O: or
Fe:° - H~ au 50~ moisture con~ent for 24 hours at RT° The tobacco was
~hon cashed wlnh 2 changes of ho~ water, d=igd and p=onein denarmined.
4. Effec= of Medium ComDosi~icn on Pr==eo!v=i= Enzo~e Seqre~an by Yea~q
A large number of yeas~s have been isolated ~urlng a survey of nhe
mic.-o~ial flora of nhe growing ~obac¢o plan: as well as from fermenn£ng
flue-cured nobacco. Many of ~hese yeas=s we=e also screened for
pro~ecl.v~ic acuivity on accoun= of our inte=esn in removing protein from
nobac:: by mic=oDiological procedures. Hewers=0 our inte~es~ resides
mainly in ~hose yeaens ~haU are a~le ~o secrete excess pronease(s) in
liqui~ media, si~=~ it is ~hese ¢ul~uras ~ha: would be useful for ~he
solubilization of pronein f=cm nobaccc.
In a Eesnri=ted screening for enzyme sac=scion, we have Ldennified two
Ha~se~u~a a~mala snrains and an unidenuifie~ yeast aurain, as possessing
~he ahiliny no secrete an acid protease in a simple, chemically-defined
medium. We also have in cur possession a cu~uure of
c~se~:!v~ica~ kindly provided b7 Dr. M.A. i~¢han=e of the ~nive=sity of
Wesne=~ On=erie, London, a~d which has been =epor=ed no he highly
proteci~uic. However, ~his 7easn does not sac=sUe prouease in ~he above
medium. We have now rscogni:ed nhau nhis yeast has ~o he provided a
comple× medium c=nuaining ~=yp~one, which i~ a panc=ea=Lc digesu of
easel=, ~ef=Ee i: will sac=sue an alkali~s ~::teasa in a liquid medium.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
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Also, monosac:harides, such as ~i;=ose a~d f=uc::se, when included in ~he
g=o~=h medlu~, shu= down enzyme sac=eti:~ by ~his yeast. Reporued herein
a:e ou~ s~uCLes on the physiological faccocs affec:i~g p:o:ease secretion
~y these ye~5:s.
P~cteclytic activity in Hanse~u!a ~ and cube= ye~s=s was evaluaued
using bovine serum albumin (BSA) and casein as ~ducer3 of p=oteasee.
Since these p=oteLns were incocpc=~ted ~n a simple mod-ium ccnuaining
glucose and ~east Niu=ogen Base (~o la~..i~a acids and (:~H,):S~,) (YNB), six
of cur yeas:s degraded both protslns, hun only H. anc~a~a and isoLaue 20a-
4 exc=eued up to 0.7 unit prouease/ml, cul~ure su~e=na:an~ within 2 days
of gr=wuh. Also, casein appeared uo be superior uo BSA as inducer of
p=o=ease by obese yeasus. C. caseLnolvu~ca deg=aded casei~ bu~ no~ BSA,
and failed :~ excreue protease in :his simple medium.
Since the hl;hly protaolyti= yeas:, C. caseino~vclca failed to produce
pc=tease in uhe simple medium used earils=, we decided ~o tssu enzyme
pc=duo:ion in a complex medium c===aini~; ¢.-yptc~e. Glucose was also
e!imi~aued f=:m ~he medium sLncs iu coui~ suppress enzi~e see=erich.
EazLie= studies had shown that whs~ ~his yeas: is gEow~ on a complex
medium such as casein age= (t:ypuc=e i=cluded), L: produces wide zones of
casei~ hydrolysis. T~us, Gn a t=ypucne-conuaini~g medium :his yeas: now
p=odu¢ed signlfican~ amoun=s of an alkaline pro:ease (0.3~ unit mL").
The
o:he~ yeasua when ~esued on this ~sdiu~, while showing p=ouein
decomposition, did no~ produce exccel!uls: pretense.
i: appears uhau R. one,ale does n=u pz¢duce exoceilula= p=cuease in a
medium conualning uryptone, while C. c~s~o~,~c~c~ needs :he lau~er for
enzyme seems:ion. To confirm these conclusions we decided ~o ~esu for
exoce!lulaz enzyme p~oducuion in ~ diffs=ent media described in Table 6.
F=om :his ~a~is i~ will be seen ~hat C. caseino~v:~c~ produces de~ecuabls
~oun=s of p==usase in medium 1 which c=nuains t=ypuonm and as additional
carbon sour=s, soluble snatch and g~ycerc!, Lnsused of glucose. This
ysas~ fails t= produce exocelluLa: pro:ease in medium Z, which is
dlffs=enu f=cm medium 1 only in ~hat glucose is used insuead of soluble
suazch and glycerol. It also failed ~o see=sue pc=:ease in nhe o~her owe
media which a=s devoid of ~rypuone and c==uain glucose. H. anomala and
isola=e 20a-¢ produce significanu amoun=s of pro~aase in medium 3, bum
none in media cos. i and 2, which connain ==yp=one. Addi=icn of 0.2%
yeast ext=ac= ~o medium 3 (medium 4) no= only slows p==uease production by
=hess :we yeasts, bun also reduces the ~=unts cf enzi.~es uluima~ely
p=cduced.
B.A.T. CONFIDENTIAL - CATEGORY I: MINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
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TABLE 6
MEDIUM COMPOSZT~O~ AN~ SECRETION OF PROTEASE BY Y~.,AST
SUPE.~ATANT PROTE~?Z - MG ML"l
(SUPE.~IATAt~T PROTZASE - UNITS HI."~)
YEAST
H. Anomala
20a-4
C. Caseinolytica
H. Anomala
20a-4
C. Caseinolytica
H. Anomala
20a-4
C. Casetnolytica
H. Anumala
20a-4
C. Caseinolyuica
Z:ICUBATZON PER~OD
0 1 2 4 7
0.90
0.90
0.36
0.90
0.92
0.92
0.94
0.94
0.90
"..00
0.96
0.9a
0.94 (Nil)
0.94 (Nil)
0.ll {0.09)
0.07 (0.04)
0.07 (0.06)
0.94 (Nil)
0.78 (Nil)
o.a2 (Nil)
0.09 (0.07)
0.17 (Nil)
O.IS (Nil)
0.84 (Nil)
0.02 (?;il)
0.03 (?Ill)
0.09 (~.09)
0.11 (:I£1)
0.07 ( :|i!}
0.54 ('zil)
0.03 (Nil)
0.02 (Nil)
0.08 (0.09)
0.06 (Nil)
0.03 (Nil)
O.S6 (Nil)
Nil (0.37)
Nil (0.44)
0.06 (Nil)
Nil (Nil)
Nil (0.07)
0.!S (Nil)
Nil (0.53)
Ni! (0.52)
0.03 (Nil)
Nil (0.18)
Nil (0.12}
0.12 (Nil)
t~il (C.S2)
Nil {C.46)
0.03 (?~il)
Ni! (~.30)
Nil (0.!9)
0.08 (Nil)
Nil (0.51)
Nil (0.51)
0.02 (Nil)
Nil (0.34)
NIL (0.28)
0.04 (Nil)
Msdaum !: YN3 0.17%; Sol. Suarcn 1.0%; Glycerol 3.0%; Tryp=one 0*~%; Casein 0.2%.
" 2: Medium i; Glucose 2.0% instead of Sol. S~arch and Glycer¢i.
" 3: YNB 0.17%; Glucose 2.0%; (NH,):SO. 0.025%; Casein 0.2%.
" 4: Medium 3 plus yeas= ex=rac= 0.2%.
An au=emp~ was made ~o increase p=o~ease secretion by yeas=s by modifying
~he culture media being used. T=yp=one and casein ¢oncnn~rauions were
increased ~o 0.75% and 2.0% respectively, and sucrose was used insuead of
glucose, C. caseino~vnica produced large amounUs of proueasa in ~he media
which conuain 0.75% :=yp=one and casein. Sucrose, unlike glucmse, did no=
suppress enzyme secretion by =his yeas=. ProUease producuion by ~his
yeas= was negligible in ~he simple medium and only a re!auively small
amounu of enzyme was de=eccad on day 7.
In ~he nex~ experimen= ~/p=one concentration was increased ~c 2.0%
(medium no. 2) and sucoose and glucose were compared for their effecu on
enzyme secretion by C. caseinolvtica. From the results of ~his
experimenu, presenusd in Table 7, it will be seen ~haU C. caseino!v~ica
elaborates large amounus of pro~ease in ~he two complex media containing
glycerol soluble e~arch or sucrose as addiuional carbon sources.
Zncreasing ~he nrypnone concenura~ion from 0.75% no 2.0% nearly doubles
~he secretion of procease (day 4 - medium 2 versus medium i). When
glucose is used as addi¢ionai carbon source, pcotease pr=duculon by C.
case£nolvtica is shu¢ down completely and prouein in ~he medium remains
unused even after I0 days (medium 3). Sucrose, on ~he ocher hand, permi:s
procsasa secreuion ccmparable ~o glycerol-scorch (medium 4). Zn a furuher
anuempu ~o increase prouease secretion by C. caseino~vti:~, ~.D% ~Eyp~one
and 3.0% sucrose were included in nhe producuion medium, whe~ 19 unius cf
pro~ease were secrsued on day 2 Isee Table 8, medium 3). This is ~he
highesu activity we have recorded since we were firs~ able ~= de~ec~
exoce!lular pronease secreuicn by ~hls ~ species, and :epresenus
abou: a SS-fold increase in enzyme produculon.
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
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cAa~z
Zrr-_c¢ oF H~nztm co~POSTT~O, oN PROZZZz~ ozcom=os:.'zo, sY YZ~STq
MEDIUM
NO.
I
3
4
YE~T
C. C~:-':oiy6~
H. An:.-..zli (0-0)
H. An:.-...~z (44-0)
S. Ros='.;s (~'xp llb-10~
Cos:.':[
C. CAs: L-.oiycim~
H. Anc..-.=L~ (0-0)
H. ,~.:.-',b (4.=-0)
S. Ros¢~= (~xp lib-lO}
Cot.u-: i
CULTURE $UPERNATANTS ,':~--STED FOR
PROTEIN CONTENT- MG ML" (PROTEASE ACTrVITY - UNUrS- ML')
INCUBATION PERIOD - DAYS
0 2 4 $ l0
1~
9.1:
11.63
I 1.38
11.38
II.00
8.80
13.00
IZ.40
12.80
12.20
0.12 (3.6l)
6.88
I 1.38
11.38
| 1.00
0.12 ($.78)
I0.7~
II.13
II.~_5
I 1.35
C. C.ls e..-.~i~ca
H. A~.o-~L~ ((NO)
H. Ano.m:zt~ (at-0)
S. Rosc:.:s (l:xp 1 lb-lO)
Con:.-.i
C. C~s,---oiyzic=
H. Anc-..~b. (0-0)
H. An.c-.~L~ (4.4-.0)
S. Rose,s ('Pxp llb-10)
Con::'d
~odLum i:
11.25 10.25
11.~-5 10.63
11."-5 11.00
10.88 0.$8 (0.01)
iI.~3 iI.25
7.13 0. I1 {4.00)
10.88 9.63
10.g8 10.88
10.75 0.gg (Ni/)
10.88 I I.P.~
0.08 (6J5)
0.70
11.38
1.30
11.00
0.10 (I'~.14)
7.63
9.:5
II.75
i 1.88
I0.00
7.00
I0.$0
1.30 (Nil]
II.00
o.~o (6.68)
$,63
10.63
0.51 (Nil)
11.75
0.0": (5.87)
0.': (Nil)
1.0.c (Nil)
0.:~ (NiJ)
I 1.38
O.OT 14.09)
3.?o (Nil)
Z..~0 (Nil)
1 I.SS
12.00
ii.00
:.35 (0.01)
8.T.c (Nil)
0.-~-~ (Nil)
0.0S (0.$8)
3.~-Z (Nil)
7.13 ,.'0.~)
0J0 (Nil)
11.25
0.~
1.60
0.T$
II.8S
0.09 (.~.32)
3.$0
1.90
IZ.O0
iZ..~O
I0..~0
~.7~
0..~
~I.~8
0.09 ','0.39)
~.00
6.~
0.10
I'(30
4:
0.17 (0.30)
12.00
0.Zl (0.3Z}
12.Y.~
O.O9 (0.09)
II.S8
YNB 0.17%; SoZ. Starch 1.0%; G~yceroL 3.0%; Try~Cone 0.75k;
CaseLn 2.0%.
Medium I, excep~ Tryptone 2.0%
" I, Glucose 3.0% instead of Sol. Starch & Glycerol
" 1, Suc=ose 3.0% .......
B.A.T. CONFIDENTIAL - CATEGORY" I: t'V[INNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
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MEDIUM
NO.
T~LE 8
PROTEASE PRODUCTION BY CANDIDA CASEZNOL~!'.$CA
C~ITURE SUPERNATANTS TESTED FOR ~ROTEIN COW,TENT - MG H.L"
(PROTEASE ACTIVITY - UNITS ML"I-
C S HR.
I0.~ 9.~0
C0.CZ) (0.06)
i0.6~ 4.7~
(0.~) (0.12)
10.~ 4.2~
(0.i:) (0.17)
INCUBAT'.ON PERIC." - DAYS
2 4 I 5 j 6 7 i 8
0.46
(0.599
0.29
(0.66)
0.18
(1.0~)
0.12
(3.84~
0.14
(i0.3~
0.2C
(!9.12)
0.i0
(7.77)
0.i!
(7.71)
0.17
(4.86;
0.10
(7.10)
0.I0
(6.23)
0.18
(1.43)
0.09
(8.~2)
0.09
(4.86)
0.1a
(0.49)
0.09
(9.45)
0.08
(S.63)
0.18
(O.S2)
0.i0
(5.42)
Medi'~ I: YN3 0.!7%; Sol. 3ua=ch 1.0%, Glycerol 3.0%; T~/p~one 0.7~%;
Casein 2.0%.
2: Medium I, excapu Tryp==ne 2.0%
3: " 2, S~crose 3% insuead of S:l. S~arch and Glycerol.
*HPA as subsuraue, i uni= = ~ 10.D. MI~'M~ ~eac~ion mixture a~ 37eC
and cH 9.0.
Since in¢rsasing ¢.-yp~cne and casein concentrations in ~he complex medium
used for C. caseino!v~c~ gave la=;e increases in prouease production by
~his yeasu, i: was decided ~o a~uemp~ to increase prousase production by
H. ancmala culuures by increasing ¢asein ¢c~:sn~raUion in uhe simple
medi'=m used f:= enzyme production by ~ese yeasts. S~crose and glucose
were also ccm;ared for :heir effec: on prouease produc=ion. The resul~s
of =his e~p.e:imenu wars a ¢ompleus surprise. The ~wo sur~ins of H.
failed ~o secrete pro~easa in naese =edia, even nhough ~hey are
known ~o do so in similar media. The only difference here was ~hau ~he
concanurauion of casein was increased ~o 2.~ and 5.0%. The results show
~hau such high concen~rauions cf casein suppress pro~ease secretion by .=..
anc~..~ia. C. caseino~Vuica, too, did sou decompose casein in ~hese simple
medla, includ&ng medium 2 concaining sucrose as addi¢i=nal carbon source.
Tha= =he high casein ccncenuraUicn suppressed prouease secretion hy8.
was ~ickly confirmed in :he sex= experiment in which casein was
inccrpora=ed au 0.2% and 2.0% concentrations. In ~his experimen~
Har~ersUein grade casein was compared wish sodium caseinaue, hcuh supplied
by I~. Both cul~ures of H. ancma!a ~es~ed, secreted significan~ amcun=s
of prDuease in the uwo media conuaining 0.2% casein and, a~ mos~, ~Eaces
of pr=¢ease in the nwo media conuaining 2.0% casein. The type of case/=
used did no~ appear Uo significanu!y influence pro~ease secretion by ~hese
yeasus. A culzure of Sz:~obo~omvz~s roseus examined in uhis experimenu
turnel out to be a poor producer cf pro~ease.
In conclusion, i~ should be poinuel DUO ~ha= we are n~w beginning ~o
idenzlfy uhe c~ndiuions under which ~hese yeasts will secreue opuimal
amounts of prczeasas in ¢he ¢ul~ure medium. Only when we fully learn ~c
conzrcl and c~=imi=e pr:=~ase production by ¢hsse yeasus, will be suc:ea~
in cur uluimaue goal of propels removal from =obacco by yeasu ~rea~men~.
B.A.T. CONFIDENTIAL - CATEGORY I: ~IINNESOTA TOBACCO LITIGATION
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BATCo document for PFSFC 1 March 1999
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3. P~oteLn Con:en: of Ci~e~:e S~oke C~densaue
Earlier protein de~e.~u, ina:i:ns in cL;areuUe smoke ¢ondensa=es showed
unreal£suic amoun=s of pEous£n suqges~ing inuerference by phenolic
substances in the pEouein ~s~e.-m. inauion procedure. S=¢h an Interference
Ls to be expected in an exu:e¢e~y l:w prouein material such as ciga:euue
smoke ¢ondensaue. In the ~:ess~u scud7, phenolic mauerial was ex~:acued
prior ~o prouein deue~minauion ~y uhe Bradford procedure. The prouein
con~en~ of condensaues fr:= 2 cigareuues, afuer repeated euhanolic
exuEacuions, is pEssenued in Table %. Yrom uhe ~a~e i~ will be seen ~hau
after 2 e~hanolio sxu:acuL:ns no fur:her removal of phenolic manet!el
occurs. Of course, i: is possible uhau large pepuide fragmenus, soluble
An e~hano!, are losn in ~h~ repea~ed exuracnian. Acuua! protein Iso!a~ion
from condensaue could ~esc!ve uhis p:cb!am.
TABLE 9
PROTE!NCONTE~ OF CIGARETTE SMOKE CO~DENSAT~S
SMOKE CONDENSATE
FROM
PLAYER'S CK 28
GAULOISES
PRO~E:N CONTENT - ~ AFTER
0.7.5
0.2.2
0.24
0..~3
0..~.=
0.35
i 2 3
ETY~OL EXTRACTIONS
0.13
0.13
0.13
0.16
0.20
0.18
0.13
0.12
0.13
0.22
0.18
0.18
Phenolic material was extracted frc~ ~he oondensates with s~hanoZ. The
pro~mins were pelleued by =entrifugauion and redissolved in 0.i M NaOH.
Protein ¢:nnenu was de~e~..i~ed using the Bradford :eagenu.
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RECORO TYPE:
SU@ TYPE:
SECURITY CODE:
FUNDI~;G BODY:
ORGA~;~ZATION:
GROUP NUHSER:
LOCAL PROJECT NUH@ER(S):
PROJECT TITLE:
PERSQ~ RESPONSTSLE:
EFFORT:
PROJECT DESCRIPTIOfT:
SCOPE:
DEPTH:
FUNCTION:
OBJECTIVE:
CLUSTER:
DATE..REVIEW WRITTE:;:
REVIEW TIT',E:
IT1 CA::A~A
41~
T-CI11
Non Rouulne A-~aly~ical ServLce ~o Suppo~
R ~ ~ Projecus and for General T=cub~eshoo~ing
POUIIN, P.; DUMONT, J.
!9~
Work under uhis projec~ is designed ~o provide
Anai~ulcal Su~o=~ for ceques~s made by
Pur=hasimg, TJchnical Secvices, Manufacturing,
Ma:keui~ and R & D. In addiuion any
mon~u3clng ~o satisfy goveEnmen~ guidelines
faii~ wiuhin uhe scope of ~his pc=je¢=.
LOC~
SUPPORT
SU~:Z~T:VE
ME~E~D DZV
Jui-! 1994
Non-Rouulne A~a!y~ical Projects ~o Sup~oE~ R&D
and • General Troubleshooting.
Pr::s~ s .=r
REVIEW TEXT: 5in:s uhe lasu review (JanuaF~ 1994) ~he
following se=vLces have been cender~d:
I. Scearm. in~ Ana!vs~s o~ Ca~o Sc~:~a~:
Cameo Special will nou be pEodu:~d any more.
2. Oual£~v ConC~=! of MenThol C~vs:~!s for Gu~Dh P~an~:
SixTy-Two (62) random s~ples of menuhol ::ys~als from 2 ehlpmen~s have
been analyzed since January 19~, amd fou~ ~o be within specifications.
3. Mo~L~o~in~ of Humec~an~ Levels !~ IT~ and OoDosi~on @~andq:
Glyce~ol levels we=e checked f:r .~R-Mac~:nald and RBH pr=duc~.s. On a
bone dry-weigh~ basis :he glyce=:! levels for 1994 were:
C~ave~ "A"
Hark Ten
Expo~ A
Expor~ A Lighu
Expo~u. A UlUra Limb:
Exporu A S?BL (11C g)
Export A Lighu SP~I (113 g)
Relvedece S~pe~o!i 200 (135 9)
Selvedere SUP.20G :: (90 g)
GLYCEROL
ls~ 2nd
QTR. QTR.
3.65 2.78
3.26 2.50
4.46 4.68
4.73 4.32
4.31 3.99
3.14 3.60
3.97 3.61
3.90 2.55
4.22 2.98
T= suppor~ d£ffe~enu pr:~ecus (T-3~1~, T-3:~4 and T-4920) ~we!vs (12)
samples, of American ci-~areuuss _n.= ~ cuu ~=~::0 have been analv=ed.
~o
deuermi~e gIyce:~l and ~r=py~en~ ~I~:=i !s~els.
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To suppc:= =he Hiddls-Eas= prc~ec= (T-3224) ~irCy-cwo {22) cigarecue
samples have ~een a=alyzed far glycer:i and propylene gi~:=l levels.
To suppc:= pr=~ec= T-5084 fi~=y-elgh= {Sai sa~les og :-~3 ==bacoo from
Corby were ues:ad f=r glycerol leve~s. Glycerol were measured Ln samples
before and af=er expansion Uo evaluaue =he glycerol losses during
expansion. The ave:age glycerol loss during expansion ;ere;
D-6C: 8.!% : S.7% (n - 19)*
* Average resul=s fc= 19 deuerminau!:~s f::m December !~i ~o May 1994.
4. Consume~ Ccm=!a~n~s ~ves~iaa~ans:
Since January 1994, specific analyses ~ere =equired for five (5) consumer
complain=3 (s~e ra=e as las~ year).
co,scaR COMPLA~TS - :~NUARY TO ~ ~99~
RESULTS
DESCR:~T~O~
SUBJEcT:YE
Oily substance
Oil spocs/3=own spots
T=bacco like glue/
Znsec~e?
T:bacco locking bad/
consumer sick
Consumer slck
Nag.
~:~ALYS IS
Mine:el o£1.
?araffL~ oiZ from
alumi~''~ foil/~cbacco
produc=~.
Moulds:':~o insects.
Moulds and bao=eria.
Old o= ¢verdriid
~obac=:.
No,-Routine Ex=~qEau=~v Work f~r Producu Oevelocmen~ or for General
T=oublssh:c=inc Ln Plants:
In January, plasticizer levels have been deuermined £n f=~: (4} samples of
fil~er rods ~o suppcr~ a production uisu. Analysis was hy ga~
chromatography using methanol as ~he ex=racuing solution, chio=ina~ed
solvents (Yreon 113 cr mechylene chlo:ide) are no longer :sad for ~his
procedure.
In March, a black fo~ rubber (insulaulng rubber) from =he RBH Dieu plan~
in Brampucn was analyzed ~o deuermine !us composition and =o assess ~he
impac= of accidence! blending w£~h ~c~acco. The manerial ~as found ~o be
an elaen:mer meaning a vulcanized syn=henlc ru~ber. Z~ ::n=ains polyvinyl
chloride as a flame reuardann, chlori~ausd paraffins, a!k~iphUhala~e,
plasuicize:s, s=abi!i:ers, eUc. This =auerlal should non ~e used in a
manner such ~ha: risk of contaminating smoklng ~obacco p::duc~s exisCs.
~n March, =he compose:ion of oily spcus found on cigareuuss made ~iCh
MaUin~e Fine Co= (5C ; pouches) was dece=m.i=ed. The oil ~as iden=ified as
Esso P=i~..cl 3S5 which is a mineral o;i havi~; no affec= c~ che ¢as=e of
smoking ¢=~accc prcdu=us. The source ~as a leak on ~he $¢P~ermunds
packing =~chine. A~:upriaUe ~c~ific~=icns have been =a~e :n ~his
=achine.
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