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t .. Accession tlu mber' 1310 Ler®dhrd Research Center creershere NITRO'SATION SUPPRESSION DURING LABORATORY FERM'.ENTATION OF PF2-1976 TOBACCO Submitted by: F. R. Perini and J. H. Br~ll Report r1Uimber: H-173 G Date, Feb. 20, 1979 Sumrnary or Abstract: Fermentation of'1976 PennsylvaniaiSeedleaf tobacco at 60% moisture in polypropylene bottles is achievedlin a laboratory fermentatidn chamber. Antioxidant phenols, ascorbates, sulfamic acid and monoethanolamine were indi- vidually tested for inhibition of nitrosaition. Only the phenols exhibited concurrent nitrosation suppression and fermentation tolerance. /lmp Xc: Dr. F. J. Schultz Dr. H . J. Minnemeyer Library

\ INTRODUCTION The work described here presents a limited number of ex- periments with PF2-1976 tobacco. The feasibility of obtaining reproducible fermentation conditiorns coupled with nitrosaition prevention in a]laboratory fermentation chamber was explored. These runs were the earliest tests for additive effectiveness in our stand'ardiized fermentation system. EXPERIMENTAL Fermentations were performed in thy-laboratory fermentation chamber disclosed in a previous report. Ground PF2-1976 tobacco (25.0.g) at 60% moisture was fermented in standardized window polypropylene bottles for four days at 45°C and 90% RH. Addi- tives were sprayed onto the tobacco in either waiter or ethanol solutions. Relative extent of fermentation was determined by leaf analysis. RESULTS AND DISCUSSION As shown in Table I, untreated, unfermented PF2-1976 ground tobacco contains 1.7 ppm of NNN. Duplicate four day fermentatiionss at 610m moisture gave an average NNN concentration of 9.1 ppm. This represents a 5.4 fold increase. Leaf analysis daita indi- cate that concomitant fermentation has taken place. Three broad compound types were tested on the tobacco: (a), antioxidant phenols, including vitamin E', (b) vitamin C and derivatives, and (c) simple basic and acidic species. Some of the additive experiiments have remained incomplete due to work ups which had been carried out with contaminated elution solvents or column ad'sorbent and with solvents of variable purity. Four PF2-1976 and eleven PF2-1977 NNN determinations were thus lost. The PF2-1977 runs were not repeated due to a change in experimentation priorit+ies. Except for the minor, if any, pH increase, leaf analyses support the contention that DL-a-tocopherol, BHT and mesitol (,2,4i„6-trirnethylphenol) do allow fermentation. All levels tested of the above additives (from 1 to 12 m w/w) also prevented or heavily suppressed NNN formation~ This is contrary to popular belief, ais expressed by Mergens, according to whom BHT and related antioxid'ants are ineffective as nitrosation blocking, agents., Our findings suggest that, the greater the vitamin E level, the greater is the extent of fermentation. Ethanol alone does retard fermentation and NNN growth. Its use with vitamin E andiBHT clouds interpretation of experi- ments~involving NNN level suppressants., On the other hand, previous reports3',4 show that both additives and ethanol con-

tribute to nitrosation inhibition. It is known that, in proper concentrations, ethanol kills most pathogenic bacteria. Some spore-bearing bacterial organisms, however, are resistant to ethanol.5 These may include some of the bacteria responsible for generating NNN in tobacco. To simplify interpretation of data involving fermentation/nitrosation suppression, ethanol will be avoided in the future. Mesitol was applied in water. The % nicotine and nitrogen are comparable to those after normal fermentation (Table I, line 2'), but the % TVB and pH': did'not change. Oxygen from the additive itself may contri- bute to the rise in C.O.D. value, as appears to have occurred with sodium ascorbate (Table II). Consequently, phenolic an- tioxidiants are being pursuedas likely NNN growth inhibitors. Ascorbates, in general, have been shown in the past to be less than ideal for nitrosation protection onitobaccol. Total inhibition of NNN formation by L-ascorbic acid (10' w/w), its sodium salt (15% w/w), and L-6-ascorbyl palmitate (5% w/w) has been confirmed, as seen in Table II. However, fer- mentation was prevented. A swaTnpingiamount of sodiumiL- ascorbate gave an anomalous result compared' to previous exper- ience. Earlier experiments withithe sodium salt exhibited' sizeable increases in NNNilevel. It may be that, at best, ascorbic acid derivatives act as antioxidants until they break down chemically and thermally during the fermentation process. From the totall information accumulated so far, use of ascor- bates on tobacco is not recommended. Although sulfamic acid did prevent NNNiformation, the leaf analysis evidence iniTable III suggests that neitherr high (initially above 9) nor low pH (initially below 5) additives are acceptable for the combined purpose of fermen- tation tolerance and nitrosaitioniprevention. Neutral derii- vatives of sulfamic acid should be considered, however. Mono- ethanolamine seems to allow some fermenting according to % nicotine drop, but a twofoldlNNNiincrease occurs. PROJECT'IONi The only general class of compounds tested so far which allows fermentation while preventing further NNN growth is the phenolic antioxidant species. Narrowing down of usable additives must be carried out onithe basis of scientific and economic efficiency, andllack of toxicity. Compounds such as BHT and analogs should be considered in particular. Changes in NNN level induced by the additive should be enhanced by increasing the NNN concentration under non-adlditiv€E laboratory fermentation conditions. Higher initial moisture (e.g.: 7'00)' and, possibly, longer fermentationitime would be ~ beneficial. ~

3 REFE RENCE S' 1 . F'., Perini and J. H. Bell, Lorillard Report Accession No. 1262' (Oct. 2'S, 1978). 2 . W. J. Mergens,, Chemical Week, 123, 24 (1978)1. . F. Perini and J. H. Bell, Progress Report on.Proj'ect H-173I,, Table II, lines 6-8 (S'ept. 9, 1977)'. 3 4 5 F. Perini and J. H. Bell, Lorillard Report Accession No. 1073, p. 6, Table I, lines 2'-4i (Nov. 29, 1977). Enjay Chemical Co., "Ethyl Alcohol", Enjay Laboratories, Publ., N.Y., N.Y., 1018 (1962:).

TABLE I A D' D I T' I V E MOISTURE L E' A F A N A L Y' S I S NNN ;ame: Concentration LEVEL, C'.O.D. g $ % i (ppm) ' mg (mmole) $ N!ic.. ; Nit. , TVB1 pH (',+10 )1 - - ^ 10.1.0' Contro 143* 2.68'* 4.03* 0.92 6.00 1.7* - - 58'.4 132* 2.41* 3.8'.5* 0.61 7.8* 9.1* 3thanol 12.5 ml ('2'12. 9 I 57'. 0 149 2.53 ' ; 4.10 1.014 5.8 2.~ 8' DL-a-Tocopherol ~ in 12.5 ml ethanol 2'.52'.5 ('0.582)' 58.01 148 2.45 3.9'3 1.01 6.3, I 2.0 " " 1250.8 (2.900) 57.0 143 2.2'3' 1.85 p.9S !'6.0 2.3 DL-a-Tocophero . in 10.0 ml I I ethanol ~3000.0 (6.965) 54.0 135* * 2'.-06 3!.54 * 0.58' 6.7* 1.8 3HT in 10.0'rnl :thanol 1535.0 (6.965) 55.9' 135* 12.07* 3.62* 0.65 6.4* 2'.5 ~iesitol 2500.0 (18.356) 58.2 163' 2.31 3.84 6.93 6.0 2.1 * Average of two runs.. TABLE II L-Ascorbic II ~ I - acid I '') 254.4 (1.444 59.0 150* 2'. 58* 3'. 92* i 11!. 12 ~ 6., 5 2.2 Sodium I L-ascorbate 37501.0' (i18.929) 54.0 163' 2.20 3'.515 0.87 5.2' 1.8 L-6-Ascorbyl palmitate in 12.5 ml ethano 1250.0 (3.0'15) 56.4 148' • 2.43 4.0'0 0.97 5.5 1.4 * Average of two runis.

TABLE III A D~~ DII T' I~ V ' E~ MOISTURE L E ~ A, P'~ A~ N A L~ Y S~~ I S, NNN Name Concentration LEVEL, C.O.D. $ mg (;mmole,)' $ Nic. Nit. TVB pH (+10 ), Monoethanol- amiine 1250.0 (20i.465), 57.7 152' 2.25 141.78 ' 0'. 9'3' '' 7. 0 3.2 Sulfamic acid 1250.0 (12.875) 57.1 141 2'.4'9 4.57 1-0 8 ! 5. 0 1.4'