Fermentation of tobacco with basidiomycetes
Y. Zhanga, M.A. Fraatza, M. Zimmera, F. Kleea, P. Seuma, B. Daubertshäusera, A. Platta, M. Di Giacomob, H. Zorna
a Institute
of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Heinrich-Buff-Ring 17, 35392 Giessen, Germany
b Philip Morris Products SA, Rue des Usines 90, 2000 Neuchâtel, Switzerland
Introduction
Workflow
Representing the highest developed class of fungi, basidiomycetes possess
as stated in the literature a unique capability of degrading some harmful and
potentially harmful constituents as well as of producing a wide range of natural
flavors. It is thus an alluring idea to reduce the levels of some harmful
compounds and to produce pleasant aroma attributes in a single fermentation
step.
Therefore, the growth of thirty basidiomycetes on four tobacco samples (fluecured tobacco strips, flue-cured stems, Burley tobacco strips, and Burley
stems) as sole carbon source was evaluated. Generation of natural aroma and
remediation of Tobacco Specific Nitrosamines (TSNA) and ammonia by
fermentation of tobacco with some basidiomycetes were performed
subsequently.
screening
analysis
fermentation
aroma
flue-cured tobacco strips
(FCT)
A
gas chromatograph with tandem mass
spectrometer and olfactory detector port
B
Burley tobacco strips
(BT)
(GC-MS/MS-O)
submerged cultures
harmful compounds
C
3d
flue-cured stems
(FS)
9d
6d
surface cultures
Burley stems
(BS)
4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK)
N'-nitrosonornicotine
(NNN)
N'-nitrosoanabasine
(NAB)
N'-nitrosoanatabine
(NAT)
sensory evaluation
raw materials
Results
1. Screening
8
7
6
6
5
5
4
4
3
3
2
2
1
1
0
0
3
FS
8
8
7
7
6
5
4
3
17
16
15
9
BS
23
II
21
19
17
15
C
D
G
H
BT
E
0.30 µg/L
17
IV
16
15
*
14
*
FCT B
C
BT
*
E
FCT B
BS
23
22
D
G
III
21
19
17
15
V
20
19
18
G
D
G
H
BT
E
BS
F
384 µg/L
16 µg/L
FCT A
FCT A
H BT E BS F
21
F
23
67 µg/L
13
13
B
25
19
20
VI
19
18
18
17
17
16
16
15
FCT A
H
VII
G
H
*
FCT D
BT
E
BS
F
5
Fig. 3 Changes of the concentrations of some odor-active compounds in tobacco fermented with
basidiomycetes and in non-fermented tobacco
4
(I 6-methyl-5-hepten-2-one; II benzaldehyde; III linalool; IV p-tolualdehyde; V -damascenone; VI -ionone; VII p-anisaldehyde;
3
* compounds detected only in the fermented tobacco)
2
1
1
0
0
6
Time (d)
6
Time (d)
6
2
3
18
FCT A
3
9
I
19
128 µg/L
25
14
9
Diameters (cm)
Diameters (cm)
9
6
Time (d)
20 µg/L
20
Liquid-liquid
extraction
(LLE)
Log2 (peak area)
0
BT
Log2 (peak area)
9
Log2 (peak area)
7
2 FCT
Headspace solid phase
microextraction
(HS-SPME)
Log2 (peak area)
9
Aroma analysis
Log2 (peak area)
4
Wolfiporia cocos
Lentinula edodes
Tyromyces chioneus
Hypsizygus tesselatus
Lepista nuda
Log2 (peak area)
6
Pleurotus sapidus
Pleurotus ostreatus
Panellus serotinus
Polyporus umbellatus
Flammulina velutipes
Log2 (peak area)
8
8
Diameters (cm)
Trametes versicolor
Pleurotus floridanus
Lentinus squarrosulus
Termitomyces albuminosus
Agrocybe aegerita
Diameters (cm)
Diameters (cm)
10
3. Analysis
9
A multitude of odor-active compounds including
3
6
Time (d)
9
Fig. 1 Growth of basidiomycetes on different types of substrates (10 g/L)
-acetylpyridine,
p-anisaldehyde,
benzaldehyde, -damascenone, 2-decanone, geranylacetone, -ionone, linalool, (Z)-linalool oxide, methyl
benzoate,
6-methyl-2-heptanone,
6-methyl-5-hepten-2-ol,
6-methyl-5-hepten-2-one,
methyl
4-
methoxybenzoate, methyl 3-methylpentanoate, methyl palmitate, methyl 2-phenylacetate, nerolidol,
2-nonanone, (Z)-3-nonen-1-ol, octanal, 1-octanol, 3-octanone, 1-octen-3-ol, 2-pentanone, 3-pentanone,
Most of the strains grew well, and FCT and FS proved to be the most
piperonal,
2-phenylacetaldehyde,
and
p-tolualdehyde
were identified by GC-MS/MS-O
promising substrates (Fig. 1). A series of volatiles, like p-anisaldehyde,
combined with HS-SPME in fermented tobacco samples. Some of the odorants
benzaldehyde, -damascenone, linalool, and 1-octen-3-ol were emitted from
generated by the fungi were semi-quantified by internal standard after LLE (Fig. 3).
the surface cultures.
Other tobacco constituents
2. Submerged fermentation
Tobacco Specific Nitrosamines (TSNA) (ng/g)
Sample
aroma mixture A
aroma mixture H
No.
NAB
NAT
NNK
NNN
Reduction (%)
NNK
Total
Ammonia
Total
N-NH3
alkaloids
(%)
(%)
FCT
< LOQ
< LOQ
19.55 ± 0.59
< LOQ
A
4.53 ± 0.17
< LOQ
0.93 ± 0.18
3.01 ± 0.08
95.2
56.7
B
7.13 ± 0.71
< LOQ
< LOQ
< LOQ
100
63.5
0.03 ± 0.000 0.73 ± 0.007
C
1.02 ± 0.16
< LOQ
2.68 ± 0.28
6.39 ± 0.58
86.3
48.4
0.020 ± 0.000 0.67 ± 0.028
D
11.96 ± 0.021
< LOQ
7.49 ± 0.18
9.45 ± 0.18
61.7
0.51
G
0.16 ± 0.007
2.78 ± 0.40
2.54 ± 0.31
10.72 ± 0.31
87.0
17.1
0.030 ± 0.000 1.05 ± 0.007
H
2.69 ± 0.22
< LOQ
2.29 ± 0.007
< LOQ
88.3
74.5
0.08 ± 0.000 0.73 ± 0.007
BT
332.6 ± 3.91
4707.13 ± 121.86
2756.15 ± 36.84
7756.94 ± 35.54
E
303.13 ± 2.09
3798.10 ± 98.44
2141.04 ± 30.58
5717.81 ± 114.05
Fig. 2 Basidiomycete/substrate combinations (for A – H cf. Fig. 3)
BS
139.78 ± 7.69
2735.35 ± 34.34
2889.25 ± 76.69
4928.00 ± 45.36
(F. pinicola = Fomitopsis pinicola; L. sulphureus = Laetiporus sulphureus)
F
109.85 ± 1.58
1432.56 ± 27.70
1994.14 ± 7.84
3688.29 ± 8.42
aroma mixture B
aroma mixture G
aroma mixture C
aroma mixture F
aroma mixture E
aroma mixture D
0.08 ± 0.007 1.37 ± 0.007
< LOQ
< LOQ
0.98 ± 0.007
0.55 ± 0.007
0.330 ± 0.000 2.26 ± 0.021
22.3
23.1
0.110 ± 0.000 2.09 ± 0.021
0.130 ± 0.000 0.87 ± 0.007
31.0
32.4
< LOQ
0.61 ± 0.021
Conclusions and outlook
Surprisingly, most of the screened basidiomycetes were able to grow well on tobacco substrates as the sole carbon and nitrogen source. Interesting aroma
impressions were perceived after fermentation. Subsequently, the responsible compounds were identified by HS-SPME combined with GC-MS/MS-O and some of
them were semi-quantified after LLE. Meanwhile, NNK, ammonia, and total alkaloids were degraded during fermentation. Taken together, fermentation of tobacco
by basidiomycetes may present an interesting option for the future improvement of tobacco derived products.