Smoke flavor enhancing agents

This invention relates to a method for enhancing the smoke flavor of a smoking material by treating the said material with an agent comprising 3-hydroxysclareol, particularly with the compound 3.beta.-hydroxysclareol. The invention further relates to the smoke-enhancing agent and a method by which it can be obtained by subjecting sclareol to a microbial transformation process. Finally it extends to smoking material incorporating the said agent.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to smoke-flavour enhancing agents, a method of 
manufacture thereof and a method of enhancing the smoke flavour of a 
smoking material. 
2. Brief Description of the Prior Art 
Flavour-enhancing agents may themselves contribute to the smoke flavour of 
a smoking material, but the present invention is more especially concerned 
with the provision of agents whose effect is to enhance the characteristic 
flavour of the smoking material itself, particularly a 
tobacco-characteristic flavour, without introducing undesirable flavour 
attributes. Apart from the evident possibility of obtaining new flavours 
or flavour tones, such agents provide an additional tool for the 
assistance of the tobacco blender whereby control for the attainment of a 
desired smoke-flavour can be exercised more reliably and/or with greater 
economy. 
Compounds chemically related to sclareol and said to be useful as additives 
to tobacco are disclosed in United Kingdom Patent Specification No. 
847,201 and U.S. Pat. No. 3,050,532. These compounds are disclosed as 
obtained by subjecting sclareol to chemical conversion processes. 
Sclareol is a diterpene compound which is obtainable from the flowers of 
the Clary sage (Salvia sclarea) and from the flowers and leaves of 
Nicotiana glutinosa. A process for recovering sclareol from Clary sage is 
disclosed in U.K. Specification No. 879,958. 
SUMMARY OF THE INVENTION 
According to the present invention, a group of compounds valuable for 
enhancing the smoke flavour of smoking material are obtained by subjecting 
sclareol to a microbial transformation process. 
Also according to the invention, a smoke-flavour enhancing agent for 
incorporation with a smoking material comprises one or more compounds from 
the group consisting of 3.alpha.-hydroxysclareol, 3.beta.-hydroxysclareol 
and 3-oxosclareol. 
The invention also provides a method of manufacture of a smoke flavouring 
agent for incorporation with a smoking material, wherein sclareol is 
contacted with a microbial culture to effect a transformation of at least 
a proportion of the sclareol to a product comprising 3-hydroxysclareol. 
The hydroxysclareol may be 3.alpha.-hydroxysclareol or 
3.beta.-hydroxysclareol, and the product may be further comprise 
3-oxosclareol. 
A product comprising these three compounds may be used as a tobacco-smoke 
flavour-enhancing agent. Alternatively, one or more of the compounds, 
advantageously being or including the more potent and higher-yielding 
3.beta.-hydroxysclareol, may be first extracted and the extracted compound 
or compounds used as tobacco-smoke flavour enhancing agent or agents. 
DETAILED DESCRIPTION OF THE INVENTION 
The invention further provides a method of enhancing the smoke flavour of a 
smoking material, wherein there is incorporated with the smoking material 
an agent comprising one or more compounds selected from the group 
consisting of 3.alpha.-hydroxysclareol, 3.beta.-hydroxysclareol and 
3-oxosclareol. The smoking material may be tobacco, a reconstituted 
tobacco material or a tobacco substitute material. Advantageously, the 
agent is or comprises 3.beta.-hydroxysclareol. The agent may be 
incorporated with a smoking material by being sprayed thereon in volatile 
solvent or by being sprinkled thereon in fine crystalline granular form. 
In the latter case a binder may be employed to bind the crystals to the 
smoking material. The agent is suitably added to the smoking material at a 
loading level within a range of 50 to 2,000 parts per million of tobacco. 
The chemical structure of 3.alpha.-hydroxysclareol may be represented as: 
##STR1## 
The chemical structure of 3.beta.-hydroxysclareol may be represented as: 
##STR2## 
The chemical structure of 3-oxosclareol may be represented as: 
##STR3## 
We have discovered that a wide range of micro-organisms, including fungi 
and bacteria, may be used in the transformation of sclareol to a product 
comprising 3.alpha.-hydroxysclareol and 3.beta.-hydroxysclareol. In some 
cases 3-oxosclareol also is produced. Of fifty fungal shake-flask cultures 
derived from a sample of air-cured leaf tobacco it was found that 
twenty-eight were effective in transforming sclareol to give 
3-hydroxysclareol, although considerable variations in extent of 
conversion were found. With each of the twenty-eight fungi 
3.beta.-hydroxysclareol was produced and with eighteen of these 
twenty-eight fungi 3.beta.-hydroxysclareol was also found to be present. 
With thirteen of these twenty-eight fungi 3-oxosclareol was found to be 
present. Seventeen of these fungi were identified as species of 
Aspergillus (8 strains), Penicillium (3 strains), Cladosporium (1 strain), 
Alternaria (1 strain), Nodulisporium (1 strain), Ulocladium (1 strain), 
Fusarium (1 strain) and Phoma (1 strain). Eleven fungi could not be 
identified due to lack of sporulation. Examples of suitable 
micro-organisms are: 
Ophiobolus herpotrichus (CBS 240.31) 
Alternaria alternata F316 (CBS 547.80) 
Cladosporium oxysporum F312 (CBS 548.80) 
Penicillium thomii F309 (CBS 549.80) 
Bacillus pumilus (NCIB 11617) 
The Ophiobolus herpotrichus is obtainable from the Centraalbureau Voor 
Schimmelcultures, Oosterstraat 1, Baarn, Holland. The culture deposits 
having the accession numbers CBS 547.80, CBS 548.80 and CBS 549.80 were 
each made with the Centraalbureau Voor Schimmelcultures on May 25, 1980. 
The respective characteristics of the fungi the subjects of these three 
deposits are given in Table 1 below. 
Culture deposit NCIB 11617 was made on Nov. 14, 1980 at the National 
Collection of Industrial Bacteria, Torry Research Station, 135 Abbey Road, 
Aberdeen, Scotland. The characteristics of the Bacillus pumilus NCIB 11617 
are: 
Morphology: Slender gram variable rods with elliptical central endospores 
not swelling the sporangium. 
Temperatures for growth: Good growth at 30.degree., 37.degree., 50.degree., 
no growth at 55.degree. C. 
Growth characteristics (nutrient agar 30.degree. C.): Colonies 1.5 mm flat, 
smooth, off-white, opaque, irregular and slightly erose. 
______________________________________ 
Biochemical characteristics: 
______________________________________ 
Catalase + 
Oxidase (Kovacs) + 
Anaerobic growth - 
Growth in 5% NaCl + 
Growth in 7% NaCl + 
Gas from glucose - 
Acetoin production + 
Casein decomposition 
+ 
Gelatine decomposition 
+ 
Starch hydrolysis - 
Nitrate reduction - 
Indole - 
Citrate - 
Arginine dehydrolase 
- 
pH in VP broth 5.2 
______________________________________ 
On the basis of these characteristics the isolate was considered to conform 
to the description of Bacillus pumilus given by Bergey's Manual of 
Determinative Bacteriology, 8th Edition (1974). 
The selection of micro-organisms which possess the property of transforming 
sclareol to yield 3.alpha.-hydroxysclareol and 3.beta.-hydroxysclareol was 
made by incubating sclareol with a range of micro-organisms obtained from 
culture collections and by direct isolation from natural substances such 
as soil and tobacco leaves. The novel products were identified by 
comparing extracts from the culture media with controls comprising culture 
media having no sclareol. The products were then isolated and purified and 
predetermined proportions thereof were incorporated with cigarette 
tobacco. 
Cigarettes filled with this tobacco were smoked by an expert panel of 
smokers to assess the effect of the respective products on the tobacco 
smoke. In all cases, smokers noted an appreciable effect upon the flavour 
of the smoke, but without the intrusion of any undesirable smoke 
attribute. In general the effect observed was an enhancement of the smoke 
flavour characteristically associated with the particular tobacco. 
The sclareol used in each of the following examples was obtained from 
commercial grade Clary sage absolute supplied by Payan & Bertrand of 
Grasse, France. The absolute was first filtered under negative pressure to 
remove liquid and the solid was then washed with a stream of ice-cold 
n-hexane at room temperature until the original green colour was totally 
removed. The remaining white solid was determined to be sclareol. 
Additional amounts of sclareol were recovered from the hexane washings by 
crystallation. The yield of sclareol averaged about 20% by weight of the 
original absolute. As will be appreciated, sclareol derived from Nicotiana 
glutinosa could equally well be used for the purpose of the present 
invention.

The following examples illustrate procedures for obtaining the sclareol 
compounds and the nature of the products obtained: 
EXAMPLE 1 
A pure culture of Ophiobolus herpotrichus CBS 240.31 was inoculated into 
two 250 ml Erlenmeyer flasks each containing 100 ml sterile malt extract 
broth of formula: 
Malt extrct: 17 g 
Mycological peptone: 3 g 
Distilled water: 1000 ml 
pH: 5.4 
The flasks were incubated on an orbital shaker at room temperature, with a 
shaker speed of 150 rpm, for seven days in order to obtain a growth of O. 
herpotrichus. 200 mg of sclareol dissolved in ethanol was then added to 
one of the two flasks and in equal quantity to a third flask containing 
100 ml of the sterile malt extract but no fungus. Incubation of all three 
flasks was continued for a further seven days after which the content of 
each flask was extracted with chloroform. After concentration, the 
extracts from the three flasks were compared with each other by means of 
thin layer chromatography (TLC), using silica gel plates and a 9:1 
chloroform/methanol solvent. For visualisation purposes, the plates were 
sprayed with a mixture of anisaldehyde and sulphuric acid and then heated. 
In the case of the extract from the flask in which sclareol was added to 
the fungus culture, the TLC procedure indicated the presence of three 
diterpene compounds additional to sclareol. These compounds were not 
present in the extracts from the other two flasks. It was thus concluded 
that the three additional diterpene compounds had resulted from a 
transformation of the sclareol brought about by the O. herpotrichus 
fungus. The three additional diterpene compounds visualised were all more 
polar than sclareol by Rf value in the TLC system: 
Compound I: Rf 0.10 
Compound II: Rf 0.17 
Compound III: Rf 0.26 
Sclareol: Rf 0.33 
Compounds I, II and III were separated by a combination of column, high 
pressure liquid and thin layer chromatography and each was then identified 
using gas chromatography-mass spectrometry, nuclear magnetic resonance and 
infra-red analysis. The following identities were established: 
Compound I: 3.alpha.-hydroxysclareol 
Compound II: 3.beta.-hydroxysclareol 
Compound III: 3-oxosclareol 
EXAMPLE 2 
O. herpotrichus was grown in 100 ml sterile malt extract according to the 
procedure of Example 1. To the culture there was then added 100 mg of 
sclareol dissolved in ethanol. At the conclusion of a further three days 
incubation period, the presence of Compounds I, II and III and residual 
sclareol was demonstrated. 
EXAMPLE 3 
The procedure of Example 2 was followed except that incubation after the 
addition of sclareol proceeded for periods of seven and eleven days. At 
the conclusion of each of these incubation periods the presence of 
Compounds I, II and III and residual sclareol was demonstrated. 
EXAMPLE 4 
The procedure of Example 2 was repeated excepting in that 400 mg of 
sclareol was employed and incubation was extended to ascertain that 
incubation period resulting in the optimum yield rate of Compounds I, II 
and III. It was observed that the development of the Compounds I, II and 
III and that of the fungus peaked at about ten to twelve days of 
incubation. 
EXAMPLE 5 
The procedure of this example was as per Example 2 but to the fungus 
culture was added 400 mg sclareol dissolved in polyoxyethylene (20) 
sorbiton monooleate, supplied by Sigma Chemicals, Poole, England under the 
trade name "Tween 80". At the conclusion of a three day incubation period 
from the addition of sclareol, Compounds I, II and III were identified as 
present, the yield of Compounds I and II amounting together to 12% by 
weight of the original sclareol. 
EXAMPLE 6 
The Example 5 procedure was repeated but with an incubation period extended 
to seven days. The yield of Compounds I and II was found to be 36% of the 
original sclareol. 
EXAMPLE 7 
The Example 5 procedure was repeated but with a ten day incubation period, 
at the conclusion of which Compounds I and II were recovered at 23% of the 
original sclareol. 
EXAMPLE 8 
O. herpotrichus was grown in a number of flasks, each containing 100 ml 
sterile malt extract broth of the formula given for Example 1, for four 
days on an orbital shaker. To each flask there was then added 200 mg 
sclareol dissolved in "Tween 80". After an incubation period of three days 
Compounds I and II were found to be present at a total weight of 110 mg, 
i.e. 55% that of the added sclareol. 
EXAMPLE 9 
The procedure was as per Example 8 but with an incubation period of seven 
days. Compounds I and II were then found to be present at a total weight 
of 134 mg, i.e. 67% that of the added sclareol. 
EXAMPLE 10 
The procedure of Example 8 was followed except that the incubation period 
was ten days, after which the total weight of the Compounds I and II was 
found to be 52 mg, i.e. 26% of the added sclareol. 
EXAMPLE 11 
2.5 l sterile malt extract broth in a 3 l fermenter vessel was innoculated 
with O. herpotrichus and incubated at 23.degree. C. with continuous 
stirring at 300 rpm and aeration. After three days 6 g sclareol dissolved 
in "Tween 80" was added. The incubation was then continued for a further 
five days, at the end of which period Compounds I and II were found to be 
present in the broth and 2 g of Compound II were recovered from the broth. 
EXAMPLE 12 
The general procedure of Example 11 was followed but a 20 l vessel 
containing 15 l sterile malt extract broth was used. 45 g sclareol in 
"Tween 80" was added and the broth stirred at 400 rpm and aerated at 5.8 
l/min. Compounds I, II and III were recovered by extraction of the liquid 
at the end of the experiment. The greater part of the unreacted sclareol 
was obtained in extraction of the fungal growth. 
EXAMPLE 13 
The procedure of Example 9 was repeated using a pure culture of Alternaria 
alternata CBS 547.80 in place of O. herpotrichus. At the conclusion of the 
seven day incubation period the Compounds I, II and III were found to be 
present and were separated from the culture medium. 
EXAMPLE 14 
The procedure followed was the same as that of Example 13 except that the 
micro-organism used was Cladosporium oxysporum CBS 548.80. Compounds I and 
II were found to be present at the end of the incubation period and were 
recovered from the culture medium. 
EXAMPLE 15 
The procedure of Example 13 was again repeated, the micro-organism this 
time used being Penicillium thomii CBS 549.80. Compounds I, II and III 
were found to be present and were separated from the culture medium. 
EXAMPLE 16 
A pure culture of Bacillus pumilus NCIB 11617 was grown in an Erlenmeyer 
flask containing 100 ml nutrient broth designated CMI and supplied by 
Oxoid Limited, Basingstoke, England. After a seven day growth period, on 
an orbital shaker at room temperature, 100 mg sclareol dissoled in 0.4 ml 
"Tween 80" was added. Incubation was then continued for a further seven 
days, after which the culture was extracted. It was shown that Compounds 
I, II and III had been formed. 
It is a particular merit of the newly identified and produced compounds 
that they enhance the tobacco-like character of the smoke from cigarettes. 
This is in contrast to many well known substances with flavour properties, 
including other derivatives of sclareol, which impart distinctive flavour 
characters, described as floral or woody, to tobacco smoke, but without 
significantly increasing the tobacco character. 
A discovery made with the new compounds is an ability to boost the natural 
tobacco smoke aroma when added to cigarette tobacco at levels between 50 
and 2000 p.p.m. and particularly between 50 and 250 p.p.m. This discovery 
is of especial value when applied to cigarettes containing a preponderance 
of air-cured tobacco and to blended cigarettes, that is cigarettes 
containing mixtures of flue-cured, air-cured and oriental tobacco in 
varying proportions. In addition, it is useful in designs of cigarettes of 
lower tar delivery in which the natural level of tobacco flavour is 
perceived as low by the smoker. 
TABLE 1 
The characteristics of the culture deposits with the accession numbers CBS 
549.80, CBS 548.80 and CBS 547.80 referred to above are tabulated below in 
respect of the following: 
I. Temperature range for growth. 
II. Growth on potato dextrose agar at 25.degree. C. 
III. Growth on malt extract agar at 25.degree. C. 
IV. Microscopic characteristics. 
CBS 549.80 Penicillium thomii 
I. Growth at 20.degree. C. and 30.degree. C. No growth at 37.degree. C. 
II. At 4 days, colony 26 mm diameter, flat granular to powdery white with 
smoke-grey areas and cream reverse. At 12 days, 65 mm diameter, some 
radial zonation, smoke-grey with white margin and buff reverse. 
III. At 4 days, white, 20 mm diameter, flat powdery to granular with 
ochreous reverse. At 12 days, 65 mm diameter, smoke-grey, white margin, 
ochreous reverse. 
IV. Abundant hard, gritty, sclerotia produced over entire surface. No 
ascomata. Conidial heads phialides, penicillate, monoverticillate. 
Condiophores smooth-walled 90-150.mu. long. Sterigmate bottle shaped 
7-12.mu., condia elliptic, smooth 1.2-2.9.mu. in long chains. 
CBS 548.80 Cladosporium oxysporum 
I. Growth at 20.degree. C. and 30.degree. C. No growth at 37.degree. C. 
II. At 4 days, 24 mm diameter, cottony to velvety growth, dark herbage 
green with dull green reverse. At 12 days, 70 mm diameter, velvety, grey 
olivaceous with paler margin and dull green reverse. 
III. At 4 days, 21 mm diameter, raised cottony to velvety, furrowed, 
glaucous-grey with cream-white margin. Reverse fuscous black with cream 
margin. At 12 days, 52 mm diameter, radially furrowed, cottony/velvet 
glaucous-grey with pale margin and fuscous black reverse. 
IV. Brown-pigmented smooth condiophores 65-95.mu. long, swollen at apex. 
Branching tree-like from apex into non-septate smooth blastospores 
2.2-4.2.mu. in branching chains `Scar` at points of detachment of 
blastospores. 
CBS 547.80 Alternaria alternata 
I. Growth at 20.degree. C. and 30.degree. C. No growth at 37.degree. C. 
II. At 14 days, 72 mm diameter, cottony growth, irregular margin, pale 
olivaceous grey with dull green reverse. 
III. At 14 days, 73 mm diameter, cottony to floccose with finely indented 
margin. Smoke-grey with reverse centre sepia shading to amber at margin. 
IV. Condia brown, muriform arising in chains from simple pigmented 
condiophores. Condia ovoid, tapering at end distal from condiphore origin, 
usually to terminal paler beak-like cell. condia 13.mu..times.21.mu..