U.S. patent application number 17/384266 was filed with the patent office on 2021-11-11 for method for treating plants with probiotics.
The applicant listed for this patent is R.J. Reynolds Tobacco Company. Invention is credited to Michael F. Davis, Jerry Wayne Marshall, Serban C. Moldoveanu, Marvin Glenn Riddick.
Application Number | 20210345658 17/384266 |
Document ID | / |
Family ID | 1000005735659 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210345658 |
Kind Code |
A1 |
Marshall; Jerry Wayne ; et
al. |
November 11, 2021 |
METHOD FOR TREATING PLANTS WITH PROBIOTICS
Abstract
A method of modifying the content of certain chemical compounds
in tobacco materials is provided, the method including treatment of
a tobacco plant component with at least one probiotic. For example,
the method may modify the asparagine content in tobacco materials,
which can result in a modification in acrylamide production when
the tobacco material is exposed to elevated temperatures. The type
of tobacco plant component treated according to the invention can
be a tobacco seed, a tobacco seedling, an immature live plant, a
mature live plant, a harvested plant, or a portion thereof.
Examples of probiotics include probiotic species of the genera
Bifidobacterium, Lactobacillus, Enterococcus, Proionobacterium,
Bacillus, Saccharomyces, Streptococcus, and mixtures thereof.
Smoking articles and other tobacco products including such
probiotic-treated tobacco materials are also provided.
Inventors: |
Marshall; Jerry Wayne;
(Stokesdale, NC) ; Moldoveanu; Serban C.;
(Winston-Salem, NC) ; Davis; Michael F.;
(Clemmons, NC) ; Riddick; Marvin Glenn;
(Winston-Salem, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
R.J. Reynolds Tobacco Company |
Winston-Salem |
NC |
US |
|
|
Family ID: |
1000005735659 |
Appl. No.: |
17/384266 |
Filed: |
July 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13444272 |
Apr 11, 2012 |
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17384266 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A24B 15/20 20130101;
A24B 15/245 20130101 |
International
Class: |
A24B 15/20 20060101
A24B015/20; A24B 15/24 20060101 A24B015/24 |
Claims
1. A method of modifying amino acid and tobacco-specific
nitrosamine content in a tobacco material, comprising contacting a
tobacco plant component with a mixture of two or more probiotics,
wherein the mixture of two or more probiotics comprises a first
probiotic selected from the genus Lactobacillus.
2. The method of claim 1, wherein the tobacco plant component is
selected from the group consisting of a tobacco seed, a tobacco
seedling, an immature live plant, a mature live plant, a harvested
plant, or a portion thereof.
3. The method of claim 1, wherein the mixture of two or more
probiotics further comprises a second probiotic selected from the
group consisting of probiotic species of the genera
Bifidobacterium, Lactobacillus, Enterococcus, Proionobacterium,
Bacillus, Saccharomyces, Streptococcus, and mixtures thereof.
4. The method of claim 1, wherein the first probiotic is are
selected from the group consisting of Lactobacillus acidophilus,
Lactobacillus alimentarius, Lactobacillus amylovorus Lactobacillus
bulgaricus, Lactobacillus bifidus, Lactobacillus brevis,
Lactobacillus casei, Lactobacillus caucasicus, Lactobacillus
crispatus, Lactobacillus curvatus, Lactobacillus delbruckii,
Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus
gasseri, Lactobacillus helveticus, Lactobacillus johnsonii,
Lactobacillus lactis, Lactobacillus leichmannii, Lactobacillus
paracasei, Lactobacillus plantarum, Lactobacillus reuteri,
Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus
sp., Lactobacillus sporogenes, and mixtures thereof.
5. The method of claim 3, wherein the second probiotic is selected
from the genus Bifidobacterium.
6. The method of claim 1, wherein the mixture of two or more
probiotics comprises two or more probiotics selected from the genus
Lactobacillus.
7. The method of claim 1, wherein the contacting step comprises
applying the mixture of two or more probiotics in a solution,
suspension, or dispersion in water.
8. The method of claim 1, wherein the asparagine content of the
tobacco material following the contacting step is reduced by at
least about 50% by weight.
9. The method of claim 1, wherein the tobacco plant component
comprises flue-cured tobacco, burley tobacco, Oriental tobacco, or
a mixture thereof.
10. The method of claim 1, further comprising incorporating the
tobacco material into a smokeless tobacco product or a smoking
article.
11. The method of claim 10, wherein the tobacco material is in the
form of cut filler.
12. The method of claim 10, wherein the tobacco material is in the
form of a tobacco blend.
13. The method of claim 10, wherein the smoking article, upon
smoking, is characterized by an acrylamide content of mainstream
smoke that is reduced relative to an untreated control smoking
article.
14. The method of claim 13, wherein the amount of acrylamide
reduction by weight in mainstream smoke is at least about 20% as
compared to an untreated control smoking article.
15. The method of claim 13, wherein the amount of acrylamide
reduction by weight in mainstream smoke is at least about 40% as
compared to an untreated control smoking article.
16. A tobacco product in the form of a cigarette or a smokeless
tobacco product prepared according to the method of claim 10.
17. A tobacco product in the form of a smoking article or a
smokeless tobacco product, the tobacco product comprising a tobacco
composition, wherein the tobacco composition comprises a
probiotic-treated tobacco material.
18. The tobacco product of claim 17, in the form of a cigarette
comprising a rod of smokable material circumscribed by a wrapping
material and a filter attached to the rod at one end thereof,
wherein the smokable material comprises a tobacco material
pre-treated with one or more probiotics to decrease the content of
asparagine.
19. The tobacco product of claim 18, wherein the smoking article,
upon smoking, is characterized by an acrylamide content of
mainstream smoke that is reduced relative to an untreated control
smoking article.
20. The tobacco product of claim 19, wherein the amount of
acrylamide reduction by weight in mainstream smoke is at least
about 20% as compared to an untreated control smoking article.
21. (canceled)
22. The method of claim 3, wherein the second probiotic is selected
from the group consisting of Bifidobacterium adolescentis,
Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium
breve, Bifidobacterium infantis, Bifidobacterium lactis,
Bifidobacterium longum, Bifidobacterium pseudocatenulatum,
Bifidobacterium pseudolongum, Bifidobacterium sp., Bifidobacterium
thermophilum, Lactococcus lactis, Streptococcus cermoris,
Streptococcus faceium, Streptococcus infantis, Streptococcus
thermophilus, Enterococcus faceium, Pediococcus acidilactici,
Staphylococcus thermophilus, Staphylococcus carnosus,
Staphylococcus xylosus, Saccharomyces boulardii, Saccharomyces
cerevisiae, Saccharomyces boulardii, Bacillus cereus var toyo,
Bacillus subtilis, Bacillus coagulans, Bacillus licheniformis, and
mixtures thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to plants and modifications to
the method of growing, harvesting, and/or treating plants (e.g.,
tobacco). Particularly, the present invention relates to products
made or derived from tobacco, or that otherwise incorporate
tobacco, and are intended for human consumption.
BACKGROUND OF THE INVENTION
[0002] Popular smoking articles, such as cigarettes, have a
substantially cylindrical rod shaped structure and include a
charge, roll or column of smokable material such as shredded
tobacco (e.g., in cut filler form) surrounded by a paper wrapper
thereby forming a so-called "tobacco rod." Normally, a cigarette
has a cylindrical filter element aligned in an end-to-end
relationship with the tobacco rod. Typically, a filter element
comprises plasticized cellulose acetate tow circumscribed by a
paper material known as "plug wrap." Certain cigarettes incorporate
a filter element having multiple segments, and one of those
segments can comprise activated charcoal particles. Typically, the
filter element is attached to one end of the tobacco rod using a
circumscribing wrapping material known as "tipping paper." It also
has become desirable to perforate the tipping material and plug
wrap, in order to provide dilution of drawn mainstream smoke with
ambient air. A cigarette is employed by a smoker by lighting one
end thereof and burning the tobacco rod. The smoker then receives
mainstream smoke into his/her mouth by drawing on the opposite end
(e.g., the filter end) of the cigarette.
[0003] The tobacco used for cigarette manufacture is typically used
in blended form. For example, certain popular tobacco blends,
commonly referred to as "American blends," comprise mixtures of
flue-cured tobacco, burley tobacco and Oriental tobacco, and in
many cases, certain processed tobaccos, such as reconstituted
tobacco and processed tobacco stems. The precise amount of each
type of tobacco within a tobacco blend used for the manufacture of
a particular cigarette brand varies from brand to brand. However,
for many tobacco blends, flue-cured tobacco makes up a relatively
large proportion of the blend, while Oriental tobacco makes up a
relatively small proportion of the blend. See, for example, Tobacco
Encyclopedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design of
Cigarettes, 3.sup.rd Ed., p. 43 (1990) and Tobacco Production,
Chemistry and Technology, Davis et al. (Eds.) p. 346 (1999).
[0004] Tobacco also may be enjoyed in a so-called "smokeless" form.
Particularly popular smokeless tobacco products are employed by
inserting some form of processed tobacco or tobacco-containing
formulation into the mouth of the user. Various types of smokeless
tobacco products are known. See for example, the types of smokeless
tobacco formulations, ingredients, and processing methodologies set
forth in U.S. Pat. No. 1,376,586 to Schwartz; U.S. Pat. No.
3,696,917 to Levi; U.S. Pat. No. 4,513,756 to Pittman et al.; U.S.
Pat. No. 4,528,993 to Sensabaugh, Jr. et al.; U.S. Pat. No.
4,624,269 to Story et al.; U.S. Pat. No. 4,991,599 to Tibbetts;
U.S. Pat. No. 4,987,907 to Townsend; U.S. Pat. No. 5,092,352 to
Sprinkle, III et al.; U.S. Pat. No. 5,387,416 to White et al.; U.S.
Pat. No. 6,668,839 to Williams; U.S. Pat. No. 6,834,654 to
Williams; U.S. Pat. No. 6,953,040 to Atchley et al.; U.S. Pat. No.
7,032,601 to Atchley et al.; and U.S. Pat. No. 7,694,686 to Atchley
et al.; US Pat. Pub. Nos. 2004/0020503 to Williams; 2005/0115580 to
Quinter et al.; 2006/0191548 to Strickland et al.; 2007/0062549 to
Holton, Jr. et al.; 2007/0186941 to Holton, Jr. et al.;
2007/0186942 to Strickland et al.; 2008/0029110 to Dube et al.;
2008/0029116 to Robinson et al.; 2008/0173317 to Robinson et al.;
2008/0196730 to Engstrom et al.; 2008/0209586 to Neilsen et al.;
2008/0305216 to Crawford et al.; 2009/0065013 to Essen et al.;
2009/0293889 to Kumar et al.; 2010/0291245 to Gao et al; and
2011/0139164 to Mua et al.; PCT WO 04/095959 to Arnarp et al. and
WO 2010/132444 to Atchley; each of which is incorporated herein by
reference.
[0005] One type of smokeless tobacco product is referred to as
"snuff." Representative types of moist snuff products, commonly
referred to as "snus," have been manufactured in Europe,
particularly in Sweden, by or through companies such as Swedish
Match AB, Fiedler & Lundgren AB, Gustavus AB, Skandinavisk
Tobakskompagni A/S, and Rocker Production AB. Snus products
available in the U.S.A. have been marketed under the tradenames
Camel Snus Frost, Camel Snus Original and Camel Snus Spice by R. J.
Reynolds Tobacco Company. See also, for example, Bryzgalov et al.,
1N1800 Life Cycle Assessment, Comparative Life Cycle Assessment of
General Loose and Portion Snus (2005). In addition, certain quality
standards associated with snus manufacture have been assembled as a
so-called GothiaTek standard. Representative smokeless tobacco
products also have been marketed under the tradenames Oliver Twist
by House of Oliver Twist A/S; Copenhagen, Skoal, SkoalDry, Rooster,
Red Seal, Husky, and Revel by U.S. Smokeless Tobacco Co.; "taboka"
by Philip Morris USA; Levi Garrett, Peachy, Taylor's Pride, Kodiak,
Hawken Wintergreen, Grizzly, Dental, Kentucky King, and Mammoth
Cave by Conwood Company, LLC; and Camel Orbs, Camel Sticks, and
Camel Strips by R. J. Reynolds Tobacco Company.
[0006] Through the years, various treatment methods and additives
have been proposed for altering the overall character or nature of
tobacco materials utilized in tobacco products. For example,
additives or treatment processes have been utilized in order to
alter the chemistry or sensory properties of the tobacco material,
or in the case of smokable tobacco materials, to alter the
chemistry or sensory properties of mainstream smoke generated by
smoking articles including the tobacco material. Various types of
bacteria and other microorganisms have been added to tobacco for
such purposes as reducing the content of certain chemical compounds
(e.g., nitrosamines including tobacco-specific nitrosamines or
"TSNAs"), nitrates, cellulosic components, and nicotine), for
example, as described in U.S. Pat. No. 4,140,136 to Geiss et al.;
U.S. Pat. No. 4,151,848 to Newton et al.; U.S. Pat. No. 4,308,877
to Mattina et al.; U.S. Pat. No. 4,476,881 to Gravely et al.; U.S.
Pat. No. 4,556,073 to Gravely et al.; U.S. Pat. No. 4,557,280 to
Gravely et al.; U.S. Pat. No. 4,566,469 to Semp et al.; U.S. Pat.
No. 5,372,149 to Roth et al.; U.S. Pat. No. 7,549,425 to Koga et
al.; U.S. Pat. No. 7,549,426 to Koga et al.; and U.S. Pat. No.
7,556,046 to Koga et al., which are all incorporated herein by
reference.
[0007] It would be desirable in the art to provide further methods
for altering the character and nature of a plant such as a tobacco
plant, as well as tobacco compositions and formulations useful in
smoking articles or smokeless tobacco products.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method of treating a plant
or a portion thereof to modify (e.g., increase and/or decrease) the
amount of certain compounds present therein. The plants to which
the method of the invention can be applied can vary, and include
without limitation any flowering plants or conifers, including
various types of vines, trees, bushes, and other plants, such as
those that bear fruit, vegetables, and legumes, as well as
grains.
[0009] In one regard, the invention involves taking a plant that is
used to produce a commodity, particularly a plant used as a source
of food or other oral products, and treating the plant to modify
the amount of certain compounds present in that part of the plant
that is harvested. Such a method may result in the part of the
plant that is harvested exhibiting certain taste changes, such as
less bitterness. Certain specific plants to which the methods can
be applied include, but are not limited to, vegetable plants such
as beans (e.g., lima beans, green beans, soy beans, coffee beans),
cabbage, okra, squash, lettuce, tomatoes, peppers, asparagus,
celery, and the like; root and bulb vegetables (e.g., radishes,
onions, garlic, and carrots); grains (e.g., wheat, barley, oats,
corn, rice, rye, sorghum); fruit-bearing plants (e.g.,
strawberries); fruit-bearing vines (e.g., grapes, melons, and
cranberries); fruit-bearing bushes (e.g., blueberries) and
fruit-bearing trees (e.g., fruits such as oranges, lemons, limes,
grapefruits, cherries, peaches, bananas, plantains, and apples);
legumes (e.g., nuts); tea; hops; and herbs and spice plants. In
certain embodiments, the method relates to tobacco.
[0010] In one aspect of the invention is provided a method for
treating a plant, comprising treating the plant with one or more
probiotics. In certain embodiments, the method can relate to
modifying (e.g., decreasing) amino acid content in plants that may
subsequently be processed into a tobacco, food or beverage product
involving the application of heat (e.g., by baking, frying, or
microwaving). Exemplary plants that may be processed into food
products with the application of heat include, but are not limited
to, cereals such as wheat and flour (e.g., for the production of
breakfast cereals, biscuits, crackers, wafers, bread, crisp bread,
and cookies), malt and barley (e.g., for the production of beer),
potatoes (e.g., for the production of potato chips and French
fries), coffee and chicory (e.g., for use in roasted coffee
beverages). For example, the levels of asparagine in certain plants
can be modified, which can result in an acrylamide content of the
food product produced therefrom that is reduced relative to an
untreated food product.
[0011] In one aspect of the invention is provided a method for
treating a tobacco plant comprising treating the tobacco plant with
one or more probiotics. For example, in certain embodiments, the
invention relates to a method of modifying (such as by decreasing)
the content of certain amino acids and tobacco-specific
nitrosamines in a tobacco material, comprising contacting a tobacco
plant component with one or more probiotics. The tobacco plant
component can vary; for example, the tobacco plant component can be
selected from the group consisting of a tobacco seed, a tobacco
seedling, an immature live plant, a mature live plant, a harvested
plant, or a portion of any of the above (e.g., a portion of a live
plant such as only the stalk or only the leaves or a portion of the
surface of the seed).
[0012] Various probiotics and mixtures thereof can be used
according to the invention. In certain embodiments, the one or more
probiotics are selected from the group consisting of probiotic
species of the genera Bifidobacterium, Lactobacillus, Enterococcus,
Proionobacterium, Bacillus, Saccharomyces, Streptococcus, and
mixtures thereof. Exemplary probiotics include, but are not limited
to, Bifidobacterium adolescentis, Bifidobacterium animalis,
Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium
infantis, Bifidobacterium lactis, Bifidobacterium longum,
Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum,
Bifidobacterium sp., Bifidobacterium thermophilum, Lactobacillus
acidophilus, Lactobacillus alimentarius, Lactobacillus amylovorus
Lactobacillus bulgaricus, Lactobacillus bifidus, Lactobacillus
brevis, Lactobacillus casei, Lactobacillus caucasicus,
Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus
delbruckii, Lactobacillus fermentum, Lactobacillus gallinarum,
Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus
johnsonii, Lactobacillus lactis, Lactobacillus leichmannii,
Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus
reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius,
Lactobacillus sp., Lactobacillus sporogenes, Lactococcus lactis,
Streptococcus cermoris, Streptococcus faceium, Streptococcus
infantis, Streptococcus thermophilus, Enterococcus faceium,
Pediococcus acidilactici, Staphylococcus thermophilus,
Staphylococcus carnosus, Staphylococcus xylosus, Saccharomyces
boulardii, Saccharomyces cerevisiae, Saccharomyces boulardii,
Bacillus cereus var toyo, Bacillus subtilis, Bacillus coagulans,
Bacillus licheniformis, and mixtures thereof.
[0013] In some embodiments, the one or more probiotics comprise at
least one probiotic selected from the genus Bifidobacterium and at
least one probiotic selected from the genus Lactobacillus. In some
embodiments, the one or more probiotics comprise two or more
probiotics selected from the genus Bifidobacterium or two or more
probiotics selected from the genus Lactobacillus.
[0014] The method of contacting the tobacco plant component with
the probiotic can vary. For example, the contacting can, in certain
embodiments, comprises applying the one or more probiotics in a
solution, suspension, or dispersion in water.
[0015] In some embodiments, the asparagine content of the tobacco
material following the step of contacting the tobacco plant
component with one or more probiotics is about 50% or less by
weight, about 40% or less, about 30% or less, or about 20% or less
than the asparagine content of tobacco plant components that have
not been contacted with a probiotic. In other words, the tobacco
component treated according to the method of the invention can
exhibit a reduction in asparagine content according to any the
percentages set forth above.
[0016] The method can, in some embodiments, further comprise the
step of incorporating the tobacco material into a smokeless tobacco
product or a smoking article. The tobacco material can be, for
example, in the form of cut filler and/or in the form of a tobacco
blend. In certain embodiments, the tobacco plant component
comprises flue-cured tobacco, burley tobacco, Oriental tobacco, or
a mixture thereof. Such a smoking article, in some embodiments,
upon smoking, is characterized by an acrylamide content of
mainstream smoke that is reduced relative to an untreated control
smoking article. The amount of acrylamide reduction by weight in
mainstream smoke can, in some embodiments, be at least about 20% as
compared to an untreated control smoking article or at least about
40% as compared to an untreated control smoking article.
[0017] In a further aspect of the invention, a tobacco product
containing a tobacco composition comprising a probiotic-treated
tobacco is provided, such as a smoking article in the form of a
cigarette or a smokeless tobacco product containing tobacco treated
according to the method of the invention. In one embodiment is
provided a smoking article in the form of a cigarette comprising a
rod of smokable material circumscribed by a wrapping material and a
filter attached to the rod at one end thereof, wherein the smokable
material comprises a tobacco material pre-treated with one or more
probiotics to modify (e.g., decrease) the content of asparagine
and, by extension, modify the content of acrylamide formed in
mainstream smoke.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to provide an understanding of embodiments of the
invention, reference is made to the appended drawings, which are
not necessarily drawn to scale, and in which reference numerals
refer to components of exemplary embodiments of the invention. The
drawings are exemplary only, and should not be construed as
limiting the invention.
[0019] FIG. 1 is an exploded perspective view of a smoking article
having the form of a cigarette, showing the smokable material, the
wrapping material components, and the filter element of the
cigarette; and
[0020] FIG. 2 is a cross-sectional view of a smokeless tobacco
product embodiment, taken across the width of the product, showing
an outer pouch filled with a smokeless tobacco composition of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention now will be described more fully
hereinafter. This invention may, however, be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art. As used in this specification and the claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Reference to "dry weight
percent" or "dry weight basis" refers to weight on the basis of dry
ingredients (i.e., all ingredients except water).
[0022] The invention provides plant materials having modified
levels of certain compounds. In one exemplary aspect, the invention
provides tobacco having modified levels of certain compounds,
tobacco products incorporating tobacco material derived from such
tobacco, and methods for preparing a tobacco having modified levels
of certain compounds and for incorporating tobacco material derived
from this tobacco within tobacco products. The method of modifying
levels of certain compounds generally comprises contacting tobacco
with one or more probiotics. It is noted that although the
discussion provided herein focuses in large part on treatment of
tobacco, a variety of other plants (including fruits, vegetables,
flowers, and components thereof) can be treated according to the
methods provided herein to afford plants and/or plant components
having modified levels of certain compounds therein.
[0023] The probiotic treatment described herein can have various
effects on the resulting tobacco material. For example, in certain
embodiments, tobacco treated with one or more probiotics exhibits
modified levels of certain chemical compounds. These changes can
result in modification of the organoleptic properties of the
tobacco, such as changes in taste or aroma (e.g., reduced
bitterness or smoother flavor). It is noted that the specific
results obtained may be related, in part, to the probiotic species
that are used in the treatment. It is believed that probiotics of
different genera, species, and/or subspecies have different effects
on the levels of various compounds within the tobacco.
[0024] In one specific embodiment, tobacco is treated with
probiotics to modify (e.g., reduce) the concentration of amino
acids and certain other components of the tobacco. Certain amino
acids and amino acid derivatives that may be modified in certain
embodiments of the present invention include, but are not limited
to, asparagine, tryptophan, oxoproline, and aspartic acid. One
exemplary amino acid that may be advantageously reduced is
asparagine. Asparagine is a precursor of acrylamide and by reducing
the levels of asparagine in tobacco, the acrylamide level in smoke
from a cigarette comprising such a tobacco can be decreased.
Additionally, reducing asparagine content in tobacco used in
smokeless tobacco products can reduce the amount of acrylamide
produced in any heat treatment process (e.g., pasteurization)
applied to the tobacco. In one embodiment, treatment of tobacco
with one or more probiotics may result in an asparagine content
that is less than about 50% by weight of that of an untreated
tobacco, less than about 40% that of an untreated tobacco, less
than about 30% that of an untreated tobacco, or less than about 20%
that of an untreated tobacco. For example, a probiotic treated
tobacco material may have between about 0% and about 70% by weight,
such as between about 5% and about 60%, and advantageously between
about 10% and about 40% the asparagine content of an untreated
tobacco material.
[0025] Correspondingly, in certain embodiment, use of a probiotic
treated tobacco can provide a smoking material that exhibits
decreased acrylamide levels in the smoke produced therefrom. For
example, in certain embodiments, the acrylamide level in the smoke
produced from a tobacco comprising 100% probiotic-treated tobacco
exhibits an acrylamide reduction of about 10% or more, about 20% or
more, about 30% or more, about 40% or more, about 50% or more,
about 60% or more by weight as compared with a control cigarette
comprising 100% tobacco that was not probiotic-treated. Further,
use of a probiotic-treated tobacco in a smokeless tobacco product
can provide a product with decreased acrylamide levels.
Probiotic-treated tobacco may be particularly useful in products
that are heat treated at any stage of processing and/or use.
[0026] In one specific embodiment, tobacco is treated with
probiotics to modify (e.g., reduce) the concentration of
tobacco-specific nitrosamines (TSNAs) in the tobacco. Exemplary
TSNA compounds include N-nitrosonornicotine (NNN),
4-methyl-N-nitrosamino-1-(3-pyridyl)-1-butanone (NNK),
N-nitrosoanatabine (NAT),
4-methyl-N-nitrosamino-1-(3-pyridyl)-1-butanol (NNAL), and
N-nitrosoanabasine (NAB). In one embodiment, treatment of tobacco
with one or more probiotics may result in a NNN content that is
less than about 60% by weight as compared to an untreated tobacco,
or less than about 50% that of an untreated tobacco. In one
embodiment, treatment of tobacco with one or more probiotics may
result in a NAT content that is less than about 70% by weight as
compared to an untreated tobacco, or less than about 60% that of an
untreated tobacco.
[0027] In certain embodiments, the levels of other compounds in
tobacco can be modified by treatment of the tobacco with
probiotics. Exemplary compounds that are reduced in some
embodiments include, but are not limited to, acylonitrile, malic
acid, quinic acid, and glucose. The decrease in these and other
compounds can vary but generally, a treated tobacco will comprise
between about 10% and about 90% by weight of each such compound as
compared with the amount of compound present in the untreated
tobacco.
[0028] As used herein, the term "probiotic" or "probiotic
microorganism" is intended to encompass all live microorganisms
that may be classified as probiotics by various sources. For
example, the Food and Agriculture Organization of the United
Nations (FAO) defines probiotics as "live microorganisms, which,
when administered in adequate amounts, confer a health benefit on
the host." In some reports, such health benefits can include, but
are not limited to: colonization of the intestinal, respiratory,
and/or urogenital tracts, cholesterol metabolism, lactose
metabolism, absorption of calcium, synthesis of vitamins, reduction
of yeast and vaginal infections, reduction of digestive problems
(e.g., constipation and diarrheal diseases), production of natural
antibiotics, lactic acid, enzymes, hydrogen peroxide, inhibition of
pathogenic microorganisms by production of antibiotic-like
substances; and a decrease in pH. Although the traditional
definition of "probiotic" relates to human and animal digestive
organisms, this term has been applied in other contexts, such as in
the field of agriculture. Certain types of probiotics and
compositional ingredients that can be added include examples set
forth in U.S. Pat. No. 8,097,245 to Harel et al.; U.S. Pat. No.
8,097,281 to Heim et al.; U.S. Pat. No. 8,101,167 to Gueniche; and
U.S. Pat. No. 8,101,170 to Plail et al., which are all incorporated
herein by reference.
[0029] It is preferred that probiotics used according to the
invention are "GRAS" (Generally Regarded as Safe), although
non-GRAS probiotics can be used in certain embodiments. Probiotics
are typically identified by their genus, species, and strain level.
Certain recognized probiotic genera include Bifidobacterium,
Lactobacillus, Enterococcus, Proionobacterium, Bacillus,
Saccharomyces, and Streptococcus. Many common probiotics are
selected from Lactobacillus species, Bifidobacterium species, and
Streptococcus thermophilus.
[0030] Exemplary probiotics include, but are not limited to,
Bifidobacterium adolescentis, Bifidobacterium animalis,
Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium
infantis, Bifidobacterium lactis, Bifidobacterium longum,
Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum,
Bifidobacterium sp., Bifidobacterium thermophilum, Lactobacillus
acidophilus, Lactobacillus alimentarius, Lactobacillus amylovorus
Lactobacillus bulgaricus, Lactobacillus bifidus, Lactobacillus
brevis, Lactobacillus casei, Lactobacillus caucasicus,
Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus
delbruckii, Lactobacillus fermentum, Lactobacillus gallinarum,
Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus
johnsonii, Lactobacillus lactis, Lactobacillus leichmannii,
Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus
reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius,
Lactobacillus sp., Lactobacillus sporogenes, Lactococcus lactis,
Streptococcus cermoris, Streptococcus faceium, Streptococcus
infantis, Streptococcus thermophilus, Enterococcus faceium,
Pediococcus acidilactici, Staphylococcus thermophilus,
Staphylococcus carnosus, Staphylococcus xylosus, Saccharomyces
boulardii, Saccharomyces cerevisiae, Saccharomyces boulardii,
Bacillus cereus var toyo, Bacillus subtilis, Bacillus coagulans,
and Bacillus licheniformis.
[0031] Advantageously, the probiotic composition used according to
the invention is a mixture of one or more probiotics. Probiotics
that may be used according to the invention may comprise blends of
one or more genus, species, and/or strain, which may, in certain
embodiments, have improved functionality as compared with a single
strain and/or species.
[0032] According to the invention, one or more probiotics can be
applied to one or more plants. In particular, they may be applied
to one or more plant components (e.g., tobacco plant components).
By "tobacco", "tobacco plant" or "tobacco plant components" is
meant tobacco at various stages of the plant life cycle. For
example, the one or more probiotics can, in certain embodiments, be
applied to a seed, seedling, unharvested plant (at varying stages
of maturity), or harvested plant, which are all considered to be
stages of the tobacco plant as described in further detail herein.
The term "plant" and "plant component" similarly relates to a plant
(e.g., a plant that produces a commodity) at various stages of the
plant life cycle. Thus, in certain embodiments, the one or more
probiotics can be applied to a seed, seedling, unharvested plant
(at varying stages of maturity), or harvested plant comprising any
type of plant, such as those described herein. The commodity
produced from that plant may comprise any portion of the plant
(e.g., the leaf, vegetable, fruit, flower, seed, stalk, or entire
plant) and thus, various portions of the plant can exhibit modified
levels of certain compounds as a result of probiotic treatment
according to the methods provided herein.
[0033] Tobacco or tobaccos to which the method provided herein is
applicable can vary. In certain embodiments, tobaccos that can be
employed include flue-cured or Virginia (e.g., K326), burley,
sun-cured (e.g., Indian Kurnool and Oriental tobaccos, including
Katerini, Prelip, Komotini, Xanthi and Yambol tobaccos), Maryland,
dark, dark-fired, dark air cured (e.g., Passanda, Cubano, Jatin and
Bezuki tobaccos), light air cured (e.g., North Wisconsin and Galpao
tobaccos), Indian air cured, Red Russian and Rustica tobaccos, as
well as various other rare or specialty tobaccos and various blends
of any of the foregoing tobaccos. Descriptions of various types of
tobaccos, growing practices and harvesting practices are set forth
in Tobacco Production, Chemistry and Technology, Davis et al.
(Eds.) (1999), which is incorporated herein by reference. Various
representative other types of plants from the Nicotiana species are
set forth in Goodspeed, The Genus Nicotiana, (Chonica Botanica)
(1954); U.S. Pat. No. 4,660,577 to Sensabaugh, Jr. et al.; U.S.
Pat. No. 5,387,416 to White et al. and U.S. Pat. No. 7,025,066 to
Lawson et al.; US Patent Appl. Pub. Nos. 2006/0037623 to Lawrence,
Jr. and 2008/0245377 to Marshall et al.; each of which is
incorporated herein by reference. Exemplary Nicotiana species
include N. tabacum, N. rustica, N. alata, N. arentsii, N.
excelsior, N. forgetiana, N. glauca, N. glutinosa, N. gossei, N.
kawakamii, N. knightiana, N. langsdorffi, N. otophora, N.
setchelli, N. sylvestris, N. tomentosa, N. tomentosiformis, N.
undulata, N. x sanderae, N. africana, N. amplexicaulis, N.
benavidesii, N. bonariensis, N. debneyi, N. longiflora, N.
maritina, N. megalosiphon, N. occidentalis, N. paniculata, N.
plumbaginifolia, N. raimondii, N. rosulata, N. simulans, N.
stocktonii, N. suaveolens, N. umbratica, N. velutina, N.
wigandioides, N. acaulis, N. acuminata, N. attenuata, N.
benthamiana, N. cavicola, N. clevelandii, N. cordifolia, N.
corymbosa, N. fragrans, N. goodspeedii, N. linearis, N. miersii, N.
nudicaulis, N. obtusifolia, N. occidentalis subsp. Hersperis, N.
pauciflora, N. petunioides, N. quadrivalvis, N. repanda, N.
rotundifolia, N. solanifolia, and N. spegazzinii.
[0034] Nicotiana species can be derived using genetic-modification
or crossbreeding techniques (e.g., tobacco plants can be
genetically engineered or crossbred to increase or decrease
production of components, characteristics or attributes). See, for
example, the types of genetic modifications of plants set forth in
U.S. Pat. No. 5,539,093 to Fitzmaurice et al.; U.S. Pat. No.
5,668,295 to Wahab et al.; U.S. Pat. No. 5,705,624 to Fitzmaurice
et al.; U.S. Pat. No. 5,844,119 to Weigl; U.S. Pat. No. 6,730,832
to Dominguez et al.; U.S. Pat. No. 7,173,170 to Liu et al.; U.S.
Pat. No. 7,208,659 to Colliver et al. and U.S. Pat. No. 7,230,160
to Benning et al.; US Patent Appl. Pub. No. 2006/0236434 to
Conkling et al.; and PCT WO 2008/103935 to Nielsen et al. See,
also, the types of tobaccos that are set forth in U.S. Pat. No.
U.S. Pat. No. 4,660,577 to Sensabaugh, Jr. et al.; U.S. Pat. No.
5,387,416 to White et al.; and U.S. Pat. No. 6,730,832 to Dominguez
et al., each of which is incorporated herein by reference. Most
preferably, the tobacco materials are those that have been
appropriately cured and aged. Especially preferred techniques and
conditions for curing flue-cured tobacco are set forth in Nestor et
al., Beitrage Tabakforsch. Int., 20 (2003) 467-475 and U.S. Pat.
No. 6,895,974 to Peele, which are incorporated herein by reference.
Representative techniques and conditions for air curing tobacco are
set forth in Roton et al., Beitrage Tabakforsch. Int., 21 (2005)
305-320 and Staaf et al., Beitrage Tabakforsch. Int., 21 (2005)
321-330, which are incorporated herein by reference. Certain types
of unusual or rare tobaccos can be sun cured. Manners and methods
for improving the smoking quality of Oriental tobaccos are set
forth in U.S. Pat. No. 7,025,066 to Lawson et al., which is
incorporated herein by reference. Representative Oriental tobaccos
include katerini, prelip, komotini, xanthi and yambol tobaccos.
Tobacco compositions including dark air cured tobacco are set forth
in US Patent Appl. Pub. No. 2008/0245377 to Marshall et al., which
is incorporated herein by reference. See also, types of tobacco as
set forth, for example, in US Patent Appl. Pub. No. 2011/0247640 to
Beeson et al., which is incorporated herein by reference.
[0035] The Nicotiana species can be selected for the content of
various compounds that are present therein. For example, in certain
embodiments, plants of the Nicotiana species (e.g., Galpao commun
tobacco) are specifically grown for their abundance of leaf surface
compounds. In certain embodiments, plants of the Nicotiana species
are specifically grown for their relatively low levels of certain
undesired compounds (e.g., asparagine). Tobacco plants can be grown
in greenhouses, growth chambers, or outdoors in fields, or grown
hydroponically.
[0036] The means by which probiotics are applied to the tobacco
plant can vary. Certain methods to treat plants with microorganisms
which could be used, or modified for use, in the present invention
are provided in U.S. Pat. No. 4,140,136 to Geiss et al.; U.S. Pat.
No. 4,151,848 to Newton et al.; U.S. Pat. No. 4,308,877 to Mattina
et al.; U.S. Pat. No. 4,476,881 to Gravely et al.; U.S. Pat. No.
4,556,073 to Gravely et al.; U.S. Pat. No. 4,557,280 to Gravely et
al.; U.S. Pat. No. 4,566,469 to Semp et al.; U.S. Pat. No.
5,372,149 to Roth et al.; U.S. Pat. No. 7,549,425 to Koga et al.;
U.S. Pat. No. 7,549,426 to Koga et al.; and U.S. Pat. No. 7,556,046
to Koga et al., all of which are incorporated herein by
reference.
[0037] The method of application of probiotics as disclosed herein
will often depend, at least in part, on the stage of the tobacco
plant. For example, in certain embodiments, the one or more
probiotics are applied to a tobacco seed prior to planting. In such
embodiments, the one or more probiotics can be applied in the form
of a seed treatment or coating. For example, the seeds can be
dipped in probiotic solution, soaked in probiotic solution, or
sprayed with probiotic solution. In certain embodiments, the one or
more probiotics are applied to a tobacco in seedling or unharvested
(live) plant form. In such embodiments, spray application of
probiotics can be used (e.g., using a hydraulic boom sprayer, air
blast sprayer, fogger, or aerial sprayer), although the method of
probiotic application is not limited thereto.
[0038] Although it may be advantageous to apply the one or more
probiotics while the tobacco plant is still in living form, it is
also possible in some embodiments to apply probiotics following
harvesting of the tobacco plants. Such application can occur at any
time following harvest, including immediately following harvest,
prior to or following post-harvest processing (e.g., drying,
curing, and/or physical processing of the plant), or at any stage
in between. The application of probiotics can be done at one stage
in the plant life cycle, or can be conducted at two or more
stages.
[0039] It can be advantageous, in some embodiments, to apply the
probiotics in liquid form (e.g., as a solution, dispersion, or
suspension). The liquid with which the probiotics is mixed can
vary, but generally, the liquid will comprise water. In some other
embodiments, the one or more probiotics can be applied dry, such as
in granule or dust form. The concentration and amount of the
probiotic used can vary. For example, in some embodiments,
probiotic is applied to plants (e.g., living plants) in a solution
comprising between about 1.times.10.sup.5 colony forming units
(CFU)/mL and about 1.times.10.sup.10 CFU/mL (e.g., about
2.times.10.sup.6 CFU/mL). CFU provides a measurement of viable
(living) cells in the probiotic sample.
[0040] In certain embodiments, other components can be applied to
the plant with the probiotics. Such components can be added within
the same formulation (e.g., solution, dispersion, suspension, or
dry form) or can be applied to the tobacco in a separate
formulation. For example, in some embodiments, one or more
surfactants are applied to the tobacco with the probiotics. The
surfactants can be, for example, non-ionic surfactants. Various
surfactants can be used, including, but not limited to, polysorbate
surfactants, such as polysorbate 20 (Tween-20.RTM.) and polysorbate
80 (Tween-80.RTM.) and poly(ethylene glycol)-based surfactants,
such as Triton.TM. Series surfactants Other reagents for helping
the probiotic coat the tobacco effectively can include various
sugars, plant extracts (e.g., yucca extracts, seaweed extracts),
and derivatives thereof.
[0041] The whole tobacco plant, or certain parts or portions of the
plant of the Nicotiana species can be used and/or treated as
provided herein. For example, virtually all of the plant (e.g., the
whole plant) can be harvested and employed as such. Alternatively,
various parts or pieces of the plant can be harvested or separated
for treatment after harvest. For example, the flower, leaves, stem,
stalk, roots, seeds, and various combinations thereof, can be
isolated for use or further treatment.
[0042] The post-harvest processing of the plant or portion thereof
can vary. After harvest, the plant, or portion thereof, can be used
in a green form (e.g., the plant or portion thereof can be used
without being subjected to any curing process). For example, the
plant or portion thereof can be used without being subjected to
significant storage, handling or processing conditions. In certain
situations, it is advantageous for the plant or portion thereof be
used virtually immediately after harvest. Alternatively, for
example, a plant or portion thereof in green form can be
refrigerated or frozen for later use, freeze dried, subjected to
irradiation, yellowed, dried, cured (e.g., using air drying
techniques or techniques that employ application of heat), heated
or cooked (e.g., roasted, fried or boiled), or otherwise subjected
to storage or treatment for later use.
[0043] The harvested plant or portion thereof can be physically
processed. The plant or portion thereof can be separated into
individual parts or pieces (e.g., the leaves can be removed from
the stems, and/or the stems and leaves can be removed from the
stalk). The harvested plant or individual parts or pieces can be
further subdivided into parts or pieces (e.g., the leaves can be
shredded, cut, comminuted, pulverized, milled or ground into pieces
or parts that can be characterized as filler-type pieces, granules,
particulates or fine powders). The tobacco material can have the
form of processed tobacco parts or pieces, cured and aged tobacco
in essentially natural lamina and/or stem form, a tobacco extract,
extracted tobacco pulp (e.g., using water as a solvent), or a
mixture of the foregoing (e.g., a mixture that combines extracted
tobacco pulp with granulated cured and aged natural tobacco
lamina). The tobacco that is used for the tobacco product most
preferably includes tobacco lamina, or a tobacco lamina and stem
mixture. Portions of the tobaccos within the tobacco product may
have processed forms, such as processed tobacco stems (e.g.,
cut-rolled stems, cut-rolled-expanded stems or cut-puffed stems),
or volume expanded tobacco (e.g., puffed tobacco, such as dry ice
expanded tobacco (DIET)). See, for example, the tobacco expansion
processes set forth in U.S. Pat. No. 4,340,073 to de la Burde et
al.; U.S. Pat. No. 5,259,403 to Guy et al.; and U.S. Pat. No.
5,908,032 to Poindexter, et al.; and U.S. Pat. No. 7,556,047 to
Poindexter, et al., all of which are incorporated by reference. In
addition, the tobacco product optionally may incorporate tobacco
that has been fermented. See, also, the types of tobacco processing
techniques set forth in PCT WO 05/063060 to Atchley et al., which
is incorporated herein by reference.
[0044] The manner by which the tobacco is provided in such forms
can vary. The plant, or parts thereof, can be subjected to external
forces or pressure (e.g., by being pressed or subjected to roll
treatment). When carrying out such processing conditions, the plant
or portion thereof can have a moisture content that approximates
its natural moisture content (e.g., its moisture content
immediately upon harvest), a moisture content achieved by adding
moisture to the plant or portion thereof, or a moisture content
that results from the drying of the plant or portion thereof. For
example, powdered, pulverized, ground or milled pieces of plants or
portions thereof can have moisture contents of less than about 25
weight percent, often less than about 20 weight percent, and
frequently less than about 15 weight percent. Tobacco parts or
pieces can be comminuted, ground or pulverized into a powder type
of form using equipment and techniques for grinding, milling, or
the like. Most preferably, the tobacco is relatively dry in form
during grinding or milling, using equipment such as hammer mills,
cutter heads, air control mills, or the like. For example, tobacco
parts or pieces may be ground or milled when the moisture content
thereof is less than about 15 weight percent to less than about 5
weight percent.
[0045] Tobacco compositions intended to be used in a smokable or
smokeless form may incorporate a single type of tobacco (e.g., in a
so-called "straight grade" form). For example, the tobacco within a
tobacco composition may be composed solely of flue-cured tobacco
(e.g., all of the tobacco may be composed, or derived from, either
flue-cured tobacco lamina or a mixture of flue-cured tobacco lamina
and flue-cured tobacco stem. The tobacco within a tobacco
composition also may have a so-called "blended" form. For example,
the tobacco within a tobacco composition of the present invention
may include a mixture of parts or pieces of flue-cured, burley
(e.g., Malawi burley tobacco) and Oriental tobaccos (e.g., as
tobacco composed of, or derived from, tobacco lamina, or a mixture
of tobacco lamina and tobacco stem). For example, a representative
blend may incorporate about 30 to about 70 parts burley tobacco
(e.g., lamina, or lamina and stem), and about 30 to about 70 parts
flue cured tobacco (e.g., stem, lamina, or lamina and stem) on a
dry weight basis. Other exemplary tobacco blends incorporate about
75 parts flue-cured tobacco, about 15 parts burley tobacco, and
about 10 parts Oriental tobacco; or about 65 parts flue-cured
tobacco, about 25 parts burley tobacco, and about 10 parts Oriental
tobacco; or about 65 parts flue-cured tobacco, about 10 parts
burley tobacco, and about 25 parts Oriental tobacco; on a dry
weight basis. Other exemplary tobacco blends incorporate about 20
to about 30 parts Oriental tobacco and about 70 to about 80 parts
flue-cured tobacco.
[0046] Tobacco that has been treated according to the present
disclosure can, in certain embodiments, be subsequently extracted.
Various extraction techniques can be used. See, for example, the
extraction processes described in US Pat. Appl. Pub. No.
2011/0247640 to Beeson et al., which is incorporated herein by
reference. Other exemplary techniques for extracting components of
tobacco are described in U.S. Pat. No. 4,144,895 to Fiore; U.S.
Pat. No. 4,150,677 to Osborne, Jr. et al.; U.S. Pat. No. 4,267,847
to Reid; U.S. Pat. No. 4,289,147 to Wildman et al.; U.S. Pat. No.
4,351,346 to Brummer et al.; U.S. Pat. No. 4,359,059 to Brummer et
al.; U.S. Pat. No. 4,506,682 to Muller; U.S. Pat. No. 4,589,428 to
Keritsis; U.S. Pat. No. 4,605,016 to Soga et al.; U.S. Pat. No.
4,716,911 to Poulose et al.; U.S. Pat. No. 4,727,889 to Niven, Jr.
et al.; U.S. Pat. No. 4,887,618 to Bernasek et al.; U.S. Pat. No.
4,941,484 to Clapp et al.; U.S. Pat. No. 4,967,771 to Fagg et al.;
U.S. Pat. No. 4,986,286 to Roberts et al.; U.S. Pat. No. 5,005,593
to Fagg et al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; U.S. Pat.
No. 5,060,669 to White et al.; U.S. Pat. No. 5,065,775 to Fagg;
U.S. Pat. No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 to
White et al.; U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat.
No. 5,131,414 to Fagg; U.S. Pat. No. 5,131,415 to Munoz et al.;
U.S. Pat. No. 5,148,819 to Fagg; U.S. Pat. No. 5,197,494 to Kramer;
U.S. Pat. No. 5,230,354 to Smith et al.; U.S. Pat. No. 5,234,008 to
Fagg; U.S. Pat. No. 5,243,999 to Smith; U.S. Pat. No. 5,301,694 to
Raymond et al.; U.S. Pat. No. 5,318,050 to Gonzalez-Parra et al.;
U.S. Pat. No. 5,343,879 to Teague; U.S. Pat. No. 5,360,022 to
Newton; U.S. Pat. No. 5,435,325 to Clapp et al.; U.S. Pat. No.
5,445,169 to Brinkley et al.; U.S. Pat. No. 6,131,584 to
Lauterbach; U.S. Pat. No. 6,298,859 to Kierulff et al.; U.S. Pat.
No. 6,772,767 to Mua et al.; and U.S. Pat. No. 7,337,782 to
Thompson, all of which are incorporated by reference herein.
[0047] The tobacco materials discussed in the present invention can
further be treated and/or processed in other ways before, after, or
during the probiotic treatment described herein. For example, if
desired, the tobacco materials can be irradiated, pasteurized, or
otherwise subjected to controlled heat treatment. Such treatment
processes are detailed, for example, in US Pat. Pub. No.
2009/0025738 to Mua et al., which is incorporated herein by
reference. In certain embodiments, tobacco materials can be treated
with water and an additive capable of inhibiting reaction of
asparagine to form acrylamide upon heating of the tobacco material
(e.g., an additive selected from the group consisting of lysine,
glycine, histidine, alanine, methionine, glutamic acid, aspartic
acid, proline, phenylalanine, valine, arginine, compositions
incorporating di- and trivalent cations, asparaginase, certain
non-reducing saccharides, certain reducing agents, phenolic
compounds, certain compounds having at least one free thiol group
or functionality, oxidizing agents, oxidation catalysts, natural
plant extracts (e.g., rosemary extract), and combinations thereof),
and combinations thereof. See, for example, the types of treatment
processes described in US Pat. Pub. Nos. 2010/0300463 and
2011/0048434 to Chen et al., and U.S. patent application Ser. No.
13/228,912, filed Sep. 9, 2011, which are all incorporated herein
by reference. In certain embodiments, this type of treatment is
useful where the original tobacco material is subjected to heat in
the extraction and/or distillation process previously described.
Although this type of treatment can be used in combination with the
probiotic treatment of the invention, it is noted that it may not
be required, as probiotic treatment can, in some embodiments,
reduce acrylamide levels to a sufficiently low level on its
own.
[0048] The probiotic-treated tobacco can be incorporated within
various types of tobacco products according to the present
invention. For example, in some embodiments, the invention provides
smoking articles, such as cigarettes, that comprise a
probiotic-treated tobacco material. Referring to FIG. 1, there is
shown a smoking article 10 in the form of a cigarette and
possessing certain representative components of a smoking article
of the present invention. The cigarette 10 includes a generally
cylindrical rod 12 of a charge or roll of smokable filler material
(e.g., about 0.3 to about 1.0 g of smokable filler material such as
probiotic-treated tobacco material) contained in a circumscribing
wrapping material 16. The rod 12 is conventionally referred to as a
"tobacco rod." The ends of the tobacco rod 12 are open to expose
the smokable filler material. The cigarette 10 is shown as having
one optional band 22 (e.g., a printed coating including a
film-forming agent, such as starch, ethylcellulose, or sodium
alginate) applied to the wrapping material 16, and that band
circumscribes the cigarette rod in a direction transverse to the
longitudinal axis of the cigarette. That is, the band 22 provides a
cross-directional region relative to the longitudinal axis of the
cigarette. The band 22 can be printed on the inner surface of the
wrapping material (i.e., facing the smokable filler material), or
less preferably, on the outer surface of the wrapping material.
Although the cigarette can possess a wrapping material having one
optional band, the cigarette also can possess wrapping material
having further optional spaced bands numbering two, three, or
more.
[0049] At one end of the tobacco rod 12 is the lighting end 18, and
at the mouth end 20 is positioned a filter element 26. The filter
element 26 positioned adjacent one end of the tobacco rod 12 such
that the filter element and tobacco rod are axially aligned in an
end-to-end relationship, preferably abutting one another. Filter
element 26 may have a generally cylindrical shape, and the diameter
thereof may be essentially equal to the diameter of the tobacco
rod. The ends of the filter element 26 permit the passage of air
and smoke therethrough.
[0050] A ventilated or air diluted smoking article can be provided
with an optional air dilution means, such as a series of
perforations 30, each of which extend through the plug wrap 28. The
optional perforations 30 can be made by various techniques known to
those of ordinary skill in the art, such as laser perforation
techniques. Alternatively, so-called off-line air dilution
techniques can be used (e.g., through the use of porous paper plug
wrap and pre-perforated tipping paper). The filter element 26 is
circumscribed along its outer circumference or longitudinal
periphery by a layer of outer plug wrap 28. During use, the smoker
lights the lighting end 18 of the cigarette 10 using a match or
cigarette lighter. As such, the smokable material 12 begins to
burn. The mouth end 20 of the cigarette 10 is placed in the lips of
the smoker. Thermal decomposition products (e.g., components of
tobacco smoke) generated by the burning smokable material 12 are
drawn through the cigarette 10, through the filter element 26, and
into the mouth of the smoker.
[0051] In certain embodiments, according to the invention, a
smoking article comprises tobacco that has been treated with
probiotics. The tobacco within the smoking article can, in some
embodiments, comprise only such probiotic-treated tobacco or can
contain varying amounts of probiotic-treated tobacco in combination
with other tobacco materials. For example, the probiotic-treated
tobacco can be present in an amount of about 25% or more, about 50%
or more, about 75% or more, about 80% or more, about 85% or more,
about 90% or more, about 95% or more, or about 100% based on the
weight of all tobacco material in the smoking article.
[0052] Referring to FIG. 2, a representative snus type of tobacco
product comprising the probiotic-treated tobacco of the present
invention is shown. In particular, FIG. 2 illustrates a smokeless
tobacco product 40 having a water-permeable outer pouch 42
containing a smokeless tobacco composition 44, wherein the tobacco
composition includes a shredded or particulate tobacco material
that has been treated with one or more probiotics. Further
additives can be admixed with, or otherwise incorporated within,
the smokeless tobacco compositions according to the invention. The
additives can be artificial, or can be obtained or derived from
herbal or biological sources. Exemplary types of additives include
salts (e.g., sodium chloride, potassium chloride, sodium citrate,
potassium citrate, sodium acetate, potassium acetate, and the
like), natural sweeteners (e.g., fructose, sucrose, glucose,
maltose, vanillin, ethylvanillin glucoside, mannose, galactose,
lactose, and the like), artificial sweeteners (e.g., sucralose,
saccharin, aspartame, acesulfame K, neotame and the like), organic
and inorganic fillers (e.g., grains, processed grains, puffed
grains, maltodextrin, dextrose, calcium carbonate, calcium
phosphate, corn starch, lactose, manitol, xylitol, sorbitol, finely
divided cellulose, and the like), binders (e.g., povidone, sodium
carboxymethylcellulose and other modified cellulosic types of
binders, sodium alginate, xanthan gum, starch-based binders, gum
arabic, lecithin, and the like), pH adjusters or buffering agents
(e.g., metal hydroxides, preferably alkali metal hydroxides such as
sodium hydroxide and potassium hydroxide, and other alkali metal
buffers such as metal carbonates, preferably potassium carbonate or
sodium carbonate, or metal bicarbonates such as sodium bicarbonate,
and the like), colorants (e.g., dyes and pigments, including
caramel coloring and titanium dioxide, and the like), humectants
(e.g., glycerin, propylene glycol, and the like), oral care
additives (e.g., thyme oil, eucalyptus oil, and zinc),
preservatives (e.g., potassium sorbate, and the like), syrups
(e.g., honey, high fructose corn syrup, and the like),
disintegration aids (e.g., microcrystalline cellulose,
croscarmellose sodium, crospovidone, sodium starch glycolate,
pregelatinized corn starch, and the like), flavorant and flavoring
mixtures, antioxidants, and mixtures thereof. If desired, the
additive can be microencapsulated as set forth in US Patent Appl.
Pub. No. 2008/0029110 to Dube et al., which is incorporated by
reference herein. In addition, exemplary encapsulated additives are
described, for example, in WO 2010/132444 A2 to Atchley, which has
been previously incorporated by reference herein.
[0053] The following examples are provided to illustrate further
the present invention, but should not be construed as limiting the
scope thereof. Unless otherwise noted, all parts and percentages
are by weight.
EXPERIMENTAL
[0054] The present invention is more fully illustrated by the
following examples, which are set forth to illustrate the present
invention and are not to be construed as limiting thereof. In the
following examples, g means gram, L means liter, mL means
milliliter, and Da means daltons. All weight percentages are
expressed on a dry basis, meaning excluding water content, unless
otherwise indicated.
Example 1
Evaluation of Burley Tobacco Following Treatment with Probiotic
Bacteria
[0055] Burley tobacco is treated prior to harvest with solutions
containing probiotic bacteria, available over the counter as a
digestive support product. Ten live burley tobacco plants are
treated with one gallon of solution containing 60.times.10.sup.9
live bacteria cells per gallon ("Senior Probiotic," from
CVS/Pharmacy.RTM., comprising Bifidobacterium bifidum,
Bifidobacterium breve, Bifidobacterium longum, Lactobacillus
acidophilus, Lactobacillus casei, Lactobacillus helveticus,
Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus
lactis, and Streptococcus thermophilus). Ten additional live burley
tobacco plants are treated with one gallon of solution containing
60.times.10.sup.9 live bacteria cells per gallon ("Super
Probiotic," from Walgreens, comprising Lactobacillus acidophilus
and Bifidobacterium lactis).
[0056] The treated burley tobacco is harvested and subjected to
curing in standard conditions. The tobacco is stalk cut and
air-cured, with the upper, middle, and lower leaves grouped and
segregated at the end of the cure. About 15 g leaf from tip,
middle, and lower part of the plant are ground and analyzed.
[0057] The treated, cured, and segregated tobacco is analyzed for
comparative levels of 63 different compounds, as well as amino acid
levels and polyphenols. Results show a reduction in the levels of
certain compounds following probiotic treatment, including amino
acids and tobacco specific acids. For example, a significant
decrease in asparagine, tryptophan, oxoproline, aspartic acid,
malic acid, quinic acid, and glucose is observed. Specifically, the
asparagine content from mid-section samples of untreated and
treated burley tobacco samples are compared; the sample treated
with "Senior Probiotic" from CVS/Pharmacy.RTM. has 35.27%
asparagine content as compared with the untreated sample and the
sample treated with "Super Probiotic" from Walgreens has 12.9%
asparagine content as compared with the untreated sample. The
levels of certain compounds increased, namely, xylitol, fructose,
galactaric acid, myoinositol, and melibiose. Each of these
compounds is present in the probiotic mixture as-purchased as a
non-active ingredient.
[0058] Similar results are noted for treated and untreated burley
tobacco samples taken from the tip parts of the plant and from the
lugs.
Example 2
Evaluation of TSNAs in Burley Tobacco Following Treatment with
Probiotic Bacteria
[0059] Burley tobacco plants are treated with two different
probiotic solutions, harvested, and cured as described in Example
1. About 15 g of leaf from the middle of the plant are ground and
analyzed for tobacco-specific nitrosamines (TSNAs). As compared
with the control, tobacco treated with "Super Probiotic" from
Walgreens contains 95% NAT and 70% NNN. As compared with a control
(untreated) tobacco, tobacco treated with Senior Probiotic from
CVS/Pharmacy.RTM. contains 57% NAT and 43% NNN.
Example 3
Evaluation of Acrylamide Content in Mainstream Smoke Produced by
Burley Tobacco Following Treatment with Probiotic Bacteria
[0060] Burley tobacco plants are treated with two different
probiotic solutions, harvested, and cured as described in Example
1. A portion of the middle stalk leaves of the treated tobacco
plants are cut and made into cigarettes. The cigarettes are tested
using a Ceruean SM 450 smoking machine (Cerulean, Linford Wood
East, MK14 6LY, United Kingdom) under ISO conditions (a 35 mL puff
volume, 2 second puff, and 60 second puff interval). Smoke is
collected in each run on a 44 mm Cambridge smoke pad. To analyze
the acrylamide content in the smoke produced from each cigarette,
the smoke pads are soaked in methanol; water is added and an
internal standard solution of .sup.2H.sub.3-acrylamide (CDN
Isotopes, Ponte-Claire, Quebec H9R1H1, Canada) is added. The
resulting extract is filtered and passed through an SFE cartridge
(Bond Elute C18), Varian, Walnut Creek, CA 9 for sample
cleanup.
[0061] The clean sample solutions are analyzed using an LC/MS/MS
technique. HPLC separation is performed on two Gemini-NX 5ul C18
150.times.2 mm columns with a guard cartridge C18 TWIN (Phenomex,
Torrance, Calif.) in series, in isocratic mode using a solvent
system of 5% methanol and 0.1% formic acid in water. The HPLC is a
1200 HPLC system (Agilent, Wilmington, Del.), with a flow rate of
0.3 mL/min and run at room temperature. The retention time observed
for .sup.2H.sub.3-acrylamide is 3.65 minutes and for acrylamide,
the retention time observed is 3.68 min.
[0062] Acrylamide is measured using an API LC/MS/MS system (AB
Sciex, MA), with an atmospheric pressure ionization electrospray in
positive ion mode MRM (multiple reaction monitoring). The
conditions include a collision cell gas 8 L/h, curtain gas 20 L/h,
ion source gas 1 50 L/h, ion source gas 2 50 L/h, ion spray voltage
5500V, temperature 400.degree. C., declustering potential 30 V,
entrance lens potential 6 V, collision cell voltage 21 V, collision
cell exit voltage 6V. The parent ion for acrylamide is m/z=72 and
for the daughter ion, m/z=55. For .sup.2H.sub.3-acrylamide, the
parent is m/z=75 and for the daughter ion, m/z=58.
[0063] As compared with a cigarette comprising control (untreated),
cigarettes comprising tobacco treated with "Super Probiotic" from
Walgreens exhibit a 29% decrease in acrylamide content in the smoke
produced therefrom. As compared with a cigarette comprising control
(untreated), cigarettes comprising tobacco treated with "Super
Probiotic" from CVS/Pharmacy.RTM. exhibit a 65% decrease in
acrylamide content in the smoke produced therefrom.
[0064] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Therefore, it is to be understood that the
invention is not to be limited to the specific embodiments
disclosed and that modifications and other embodiments are intended
to be included within the scope of the appended claims.
[0065] Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation.
* * * * *