U.S. patent application number 13/972303 was filed with the patent office on 2013-12-19 for fermentation process.
This patent application is currently assigned to Givaudan SA. The applicant listed for this patent is Givaudan SA. Invention is credited to Eva Christina Maria Binggeli, Thomas Muench.
Application Number | 20130337114 13/972303 |
Document ID | / |
Family ID | 41786102 |
Filed Date | 2013-12-19 |
United States Patent
Application |
20130337114 |
Kind Code |
A1 |
Binggeli; Eva Christina Maria ;
et al. |
December 19, 2013 |
Fermentation Process
Abstract
Provided is a fermentation process wherein green ripe uncured
vanilla beans are incubated with [microbes] bacillus subtilis in
isolated form to convert glucovanillin to vanillin while
consistently forming a fully developed well-balanced vanilla aroma
without off-notes.
Inventors: |
Binggeli; Eva Christina Maria;
(Mannedorf, CH) ; Muench; Thomas; (Illnau,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Givaudan SA |
Vernier |
|
CH |
|
|
Assignee: |
Givaudan SA
Vernier
CH
|
Family ID: |
41786102 |
Appl. No.: |
13/972303 |
Filed: |
August 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13132978 |
Jun 6, 2011 |
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PCT/CH2009/000393 |
Dec 11, 2009 |
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13972303 |
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61122028 |
Dec 12, 2008 |
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Current U.S.
Class: |
426/44 ; 426/650;
435/252.5 |
Current CPC
Class: |
C12P 7/24 20130101; C12R
1/125 20130101; A23L 27/12 20160801 |
Class at
Publication: |
426/44 ; 426/650;
435/252.5 |
International
Class: |
A23L 1/222 20060101
A23L001/222 |
Claims
1. A process of fermenting vanilla beans the process comprising the
step of: incubating uncured vanilla beans with Bacillus subtilis in
isolated form, wherein the incubation with B. subtilis lasts for
about 24 hours to about 120 hours.
2. The process of claim 1 wherein the incubation with B. subtilis
is performed at a temperature from about 40.degree. C. to about
65.degree. C.
3. The process of claim 1 wherein the uncured vanilla beans are
dried and reconstituted with water prior to the step of incubation
with B. subtilis.
4. The process of claim 1 wherein before incubation with B.
subtilis, the uncured vanilla beans are washed.
5. The process of claim 1, wherein after the incubation with B.
subtilis, one or more enzymes comprising a beta-glucosidase enzyme
are added.
6. The process of claim 1, wherein after the incubation with B.
subtilis, the vanilla beans are dried to a low moisture of about
30% to 20% at a temperature from about 20.degree. C. to about
60.degree. C.
7. The process claim 1 wherein a solvent is added to the resulting
product to form a vanilla extract.
8. The process of claim 1 wherein the incubation with B. subtilis
is performed in a vessel selected from the group consisting of a
sweating tank wherein the vanilla beans are stationary, and a
stirred tank.
9. The process of claim 8 wherein the incubation with B. subtilis
is performed in a closed system of a sweating tank wherein the
vanilla beans are stationary and wherein the exudate from the
vanilla beans is being recycled.
10. The process of claim 1 wherein the incubation with B. subtilis
is performed in an open system.
11. The process of claim 1, wherein B. subtilis is selected from
the group consisting of B. subtilis subtilis, a wildtype B.
subtilis strain isolated from vanilla beans, and B. subtilis
subtilis strain NRRL B-50118.
12. (canceled)
13. A process according to claim 1, wherein B. subtilis used in the
fermentation step is provided from a stock culture or as a
pre-culture.
14. (canceled)
15. B. subtilis subtilis strain NRRL B-50118.
16. A flavoring product comprising a fermented vanilla bean product
produced from the process according to claim 1.
17. A flavoring product according to claim 16, wherein the
fermented vanilla bean product is selected from the group
consisting of vanilla pods, partially comminuted vanilla pods,
comminuted vanilla pods, ground vanilla pod powder, vanilla
extract.
18. An ingestible composition comprising a fermented bean product
produced from the process according to claim 1 is provided.
19. A process for producing a vanilla extract, the process
comprising the steps of: a) optionally, washing vanilla beans prior
to any subsequent process step; b) inoculating fresh, dried or
reconstituted vanilla beans with Bacillus subtilis in isolated
form, and incubating the inoculated vanilla beans for about 24
hours to about 120 hours.
20. A process according to claim 19, comprising the further step
of: cutting the vanilla beans prior to or subsequent to inoculation
with Bacillus subtilis.
21. A process according to claim 19, comprising the further step
of: adding one or more enzymes comprising a beta-glucosidase enzyme
after the incubation with B. subtilis.
22. A process according to claim 19, wherein the incubation of the
inoculated vanilla beans is at a temperature in the range of about
40.degree. C. to about 65.degree. C.
23. A process according to claim 19, comprising the further step
of: subsequent to b) incubating the inoculated vanilla beans, c)
drying the vanilla beans to a moisture content about 25%-30%
w/w.
24. A process according to claim 24, comprising the further step
of: subsequent to c) drying the inoculated vanilla beans, d)
extracting the vanilla beans with a solvent.
25. The process according to claim 19, wherein the Bacillus
subtilis is selected from the group consisting of: B. subtilis
subtilis, a wildtype B. subtilis strain isolated from vanilla
beans, and B. subtilis subtilis strain NRRL B-50118.
26. The process according to claim 19, wherein the B. subtilis used
in the fermentation step is provided from a stock culture or as a
pre-culture.
27. A flavoring product comprising a vanilla extract produced from
the process according to claim 19.
28. A flavoring product according to claim 27, wherein the product
is selected from the group consisting of vanilla pods, partially
comminuted vanilla pods, comminuted vanilla pods, ground vanilla
pod powder, vanilla extract.
29. An ingestible composition comprising a vanilla extract produced
from the process according to claim 19.
30. A method of using a flavoring product according to claim 16 as
a flavoring composition.
31. A method of using a flavoring product according to claim 27 as
a flavoring composition.
32. A process of fermenting vanilla beans, the process comprising
the steps of: washing uncured vanilla beans and subsequently,
incubating uncured vanilla beans with Bacillus subtilis in isolated
form, wherein the incubation with B. subtilis lasts for about 24
hours to about 120 hours.
33. The process according to claim 32 wherein the uncured vanilla
beans are washed in water wherein the water is at a temperature in
the range of about 20.degree. C. to about 85.degree. C.
34. A process according to claim 32, wherein the water is is at a
temperature in the range of about 60.degree. C. to about 85.degree.
C.
35. A process according to claim 32, wherein the uncured vanilla
beans are washed in an aqueous composition comprising alcohol.
Description
TECHNICAL FIELD
[0001] Disclosed is a fermentation process employing Bacillus
subtilis wherein green (ripe but uncured) vanilla beans are
incubated with B. subtilis to convert glucovanillin to vanillin,
and to convert other precursor vanilla flavor compounds bound to
glucose to the relevant non-glucose-bound vanilla flavor
compound.
BACKGROUND
[0002] Vanilla extracts comprising vanillin and other vanilla
flavor compounds are produced by subjecting ripe green vanilla
beans to a curing and extraction process. The curing process
releases vanillin from glucovanillin and forms the brown-black
whole vanilla beans commercially available. The cured beans can
then be extracted to form a liquid vanilla extract.
[0003] The curing period usually lasts many weeks or months,
involves repeated quality controls and depends on the environmental
conditions. While the sensory profile/flavor of the resulting
extracts can be excellent, even under optimal conditions the
vanillin yield is very low and usually up to about 2.2% per dry
weight of vanilla beans. Shorter curing and extraction processes
known in the art either result in a similarly low average vanillin
yield, and/or the sensory profile is not fully developed/less
complex with an unbalanced flavor profile, and/or contains
off-notes. One example of such a process is in U.S. Pat. No.
3,663,238, which describes a curing/drying process for green
vanilla beans in a sweating tank that involves cutting green beans
and incubating them at 140.degree. F. (60.degree. C.) for 70-78
hours and drying them with forced air at the same temperature to a
moisture content of 35-40%, and at room temperature to a final
moisture content of 20-25%.
[0004] The long curing period of traditional processes employs
naturally occurring enzymatic reactions. The enzymatic reactions in
the vanilla beans from its precursor glucovanillin to vanillin are
believed to be mainly caused by residual plant enzymes, in
particular glucosidase enzymes.
[0005] Notably, B. subtilis was not previously known to be involved
in or useful for the production of vanilla flavour compounds in
vanilla beans. The bacterium was known merely for being capable of
expressing various enzymes, including, among many others, the
b-glucosidase enzyme.
SUMMARY
[0006] Provided is a fermentation process to produce vanilla flavor
compounds, wherein green ripe uncured vanilla beans are incubated
with B. subtilis from a stock culture or with pre-cultured B.
subtilis.
[0007] The use of B. subtilis in a process to produce vanillin from
glucovanillin and to convert other precursor vanilla flavor
compounds bound to glucose to the relevant non-glucose-bound
vanilla flavor compound (including, without limitation, vanillic
acid, p-hydroxybenzaldehyde, and p-hydroxynbenzoic acid) as
described herein produces a comparable or higher yield than
traditional processes but with a more consistent fully developed
complex sensory profile and without off-notes. Off-notes often
present in traditional processes, without wishing to be bound by
theory, appear to be caused at least in part by various bacteria
and fungi that are present on or in the vanilla beans. The addition
of B. subtilis reduces or avoid these off-notes. The effect can be
even more improved by reducing the microorganism count prior to the
addition of B. subtilis.
Provided is the following: [0008] (1) A process of fermenting
vanilla beans comprising the incubation of uncured vanilla beans
with Bacillus subtilis in isolated form, wherein the incubation
with B. subtilis lasts for about 24 hours to about 120 hours.
[0009] For example, without limitation, B. subtilis can be added to
the vanilla beans from a stock culture of B. subtilis, or as a
pre-culture of B. subtilis. [0010] (2) The process as described
herein, including under (1), wherein the incubation with B.
subtilis is performed at a temperature from about 40.degree. C. to
about 65.degree. C. [0011] (3) The process as described herein,
including under any one of (1) and (2), wherein the uncured vanilla
beans are dried and reconstituted with water previous to the
incubation with B. subtilis. [0012] (4) The process as described
herein, including under any one of (1) to (3), wherein before
incubation with B. subtilis, the uncured vanilla beans are washed.
[0013] (5) The process as described herein, including under any one
of (1) to (4), wherein after the incubation with B. subtilis, one
or more enzymes comprising a beta-glucosidase enzyme are added.
[0014] (6) The process as described herein, including under any one
of (1) to (5), wherein after the incubation with B. subtilis, the
vanilla beans are dried to a low moisture of about 30% to 20% at
temperature from about 20.degree. C. to about 60.degree. C. [0015]
(7) The process as described herein, including under any one of (1)
to (6), wherein a solvent is added to the resulting product to form
a vanilla extract. [0016] (8) The process as described herein,
including under any one of (1) to (7), wherein the incubation with
B. subtilis is performed in a vessel selected from the group
consisting of a sweating tank wherein the vanilla beans are
stationary, and a stirred tank. [0017] (9) The process as described
herein, including under (8), wherein the incubation with B.
subtilis is performed in a closed system of a sweating tank wherein
the vanilla beans are stationary and wherein the exudate from the
vanilla beans is being recycled. [0018] (10) The process as
described herein, including under any one of (1) to (8), wherein
the incubation with B. subtilis is performed in an open system.
[0019] (11) The process as described herein, including under any
one of (1) to (10), wherein B. subtilis is selected from the group
consisting of B. subtilis subtilis, a wildtype B. subtilis strain
isolated from vanilla beans, and B. subtilis subtilis strain NRRL
B-50118. [0020] (12) A fermented vanilla bean product wherein in
its fermentation B. subtilis was provided in isolated form. [0021]
(13) The fermented vanilla bean product as described herein,
including under (13), wherein B. subtilis was provided from a stock
culture or as a pre-culture. [0022] (14) The fermented vanilla bean
product as described herein, including under (12), selected from
the group consisting of vanilla pods, partially comminuted vanilla
pods, comminuted vanilla pods, ground vanilla pod powder, vanilla
extract. [0023] (15) The B. subtilis subtilis strain NRRL
B-50118.
DETAILED DESCRIPTION
[0024] Applicant discovered that incubation with B. subtilis can be
used to ensure a good vanillin yield and provide a consistently
high quality well-balanced complex vanilla flavor profile without
off-notes in the resulting vanilla beans and their extracts.
Without wishing to be bound by theory, applicant believes that this
is due to the microbial enzymes converting glucovanillin to
vanillin (and other gluco-precursor vanilla flavor compounds bound
to glucose to the relevant non-glucose-bound vanilla flavor
compound) to a significant extent, instead of, or in addition to,
the plant enzymes. Again, without wishing to be bound by theory,
the potential antimicrobial, antiviral and antifungal properties of
B. subtilis on the variety of microorganisms naturally occurring on
or in vanilla beans may contribute to reduce or avoid the formation
of off-notes during fermentation. Alternatively or additionally,
the growth of B. subtilis may be so fast in comparison to other
naturally present microorganisms that it deprives them of their
necessary substrate. Furthermore, B. subtilis is a very adaptable
hardy microorganism that requires little and will thrive in many
minimal media under almost all conditions, including aerob and
anaerob conditions.
[0025] Vanilla beans are incubated with B. subtilis under
conditions that allow for bacterial growth until the desired degree
of degradation of glucovanillin to vanillin and other vanilla
flavor compounds from their gluco-precursors is achieved. To form
an extract, a solvent may be added. The process is described in
detail below.
[0026] There are currently three major cultivars of the genus
vanilla grown globally, all derived from a species originally found
in Mesoamerica: Vanilla planifolia (V. planifolia, syn. V.
fragrans), grown on Madagascar, Reunion and other tropical areas
along the Indian Ocean; V. tahitensis, grown in the South Pacific;
and V. pompona, found in the West Indies, Central and South
America. The majority of the world's vanilla that is produced is
the V. planifolia variety, grown in a small region of the east
African nation of Madagascar and Indonesia, and which when produced
in Madagascar is more commonly known as "Madagascar-Bourbon"
vanilla.
[0027] Vanilla spec. include, without limitation, V. planifolia, V.
tahitensis, V. pompona, and hybrids of these or other vanilla spec.
plants, for example, without limitation, hybrids of V. planifolia
and V. pompona.
[0028] Vanilla beans require about 6 to 9 months to fully develop
on the vine, during which stage the beans are green in color. At
the end of the growing stage the beans are still green with a small
yellow tip and sometimes referred to as "blossom-end yellow" or
"mature", which is the stage at which ripe green beans are
harvested. When traditionally cured, they turn from green to brown.
A green vanilla bean therefore is a mature uncured bean.
[0029] The vanilla beans incubated with B. subtilis are uncured
beans, i.e. beans that have not yet been subjected to a process to
release vanillin from glucovanillin. The uncured beans may be fresh
green beans, or "green" (uncured) beans that have been dried and
then reconstituted with water (which are brown in color). The
latter usually provide a lower vanillin yield when compared to
green beans that have not been dried and reconstituted. The vanilla
beans may be used as whole beans, or in comminuted or ground form.
The vanilla beans may be comminuted by any suitable means to render
them to pieces, for example, without limitation, about 1-2 cm long.
Any suitable tool or mechanism may be employed. For example, the
beans may be cut or chopped. The cuts generally proceed
transversely of the long axes of the beans though other directions
are possible as well.
[0030] Optionally, the beans are washed prior to incubation with B.
subtilis (thereby reducing the initial microbial count and reducing
unwanted microbes) that may naturally occur on the beans. The
washing of beans may be performed in water, in water and detergent,
or in an aqueous alcohol solution, and at a high enough temperature
for a long enough time to reduce the microbial count. For example,
washing may be performed in water and detergent at ambient
temperature (about 20-30.degree. C.), or in hot water (about
60-85.degree. C., depending on length of exposure, for example,
about 1 min at about 85.degree. C., or about 5 min at about
60.degree. C.), or in 70% alcohol for about 5 to 10 min.
[0031] For microbial incubation of the vanilla beans, Bacillus
subtilis (B. subtilis) is used. B. subtilis is a very well known
and well characterized gram positive bacterium commonly found in
soil. It usually grows aerobic, but can grow anaerobically as well.
B. subtilis provides a very good vanillin yield that other Bacillus
bacteria appear not to be able to achieve. An example of a very
suitable B. subtilis subspecies is B. subtilis subtilis, and B.
subtilis strain NRRL B-50118, deposited Mar. 20, 2008 with the ARS
Patent Culture Collection.
[0032] Other B. subtilis subtilis or B. subtilis strains, in
particular, for regulatory reasons, wild type strains isolated from
vanilla beans, may be used. Primary isolations of wildtype strains
can be performed as is well known in the art, for example on
nutrient agar (peptone 5 g/l, beef extract 3 g/l, agar 15 g/l,
pH6.8) or plates of J-agar (tryptone 5 g/l, yeast extract 15 g/l,
K2HPO4 3 g/l, glucose 2 g/l, agar 20 g/l, pH 7.4).
[0033] Various suitable growth media suitable for B. subtilis are
well known in the art. B. subtilis has little requirements for its
growth conditions and media, it grows in a minimal defined medium
with no added growth factors. A. Demain (J Bacteriol. 1958 May;
75(5): 517-522) describes some examples of constituents suitable
minimal media should include. Minimal media are particularly useful
to isolate wildtype strains. For culture, usually nutrient media
are used. Nutrient media contain a carbon source (for example,
without limitation, glucose), water, various salts, and a source of
aminoacids/nitrogen. Nutrient agar and nutrient broth (for example,
comprising as aminoacid/nitrogen source peptone and meat extract,
at a neutral pH of about 7) are suitable to culture B. subtilis,
for example, LB medium is a suitable standard medium. A standard
culture temperature is about 30.degree. C. to about 37.degree.
C.
[0034] For a B. subtilis pre-culture, any suitable standard medium
may be used.
[0035] B. subtilis may be cultured according to well known culture
conditions at a suitable temperature, pH and moisture content.
Suitable conditions include, without limitation, about 25.degree.
C. to about 60.degree. C., with a temperature optimum at about
37.degree. C., a pH of about 4.5 to about 7.5, for example, without
limitation, pH 5.5 to 6.5, a moisture content of 55% or higher, and
optionally a medium to adjust osmotic pressure. When incubated with
the vanilla beans, the same conditions apply except for a higher
culture temperature from about 45.degree. C. to about 65.degree.
C., for example, without limitation, from about 45.degree. to about
60.degree. C., or from about 50.degree. C. to about 60.degree.
C.
[0036] Optionally, oxygen may be added/sufficient aeration may be
provided, for example, without limitation, by a fan, to reduce
growth of unwanted anaerobic bacteria.
[0037] Incubation of the vanilla beans with B. subtilis can be done
in any suitable environment or vessel, for example, without
limitation, in a tank or sweating tank, for example as described in
U.S. Pat. No. 3,663,238,
[0038] The exudate ("sweat") of the vanilla beans, for example in a
sweating tank, may optionally be recycled; alternatively, an open
system may be used.
[0039] The container may optionally be stirred to provide mixing
and/or aeration.
[0040] For example, in U.S. Pat. No. 3,663,238, a stainless steel
vessel is equipped with stacked perforated trays and provided a
temperature control and a closed exudate recirculation system. The
exudate ("sweat" of the heated beans) is collected and may contain
bacteria derived from the surface of the beans. The exudate flows
through the perforations/holes in the trays through all stacked
trays to an outlet at the bottom of the vessel, and is collected
and reintroduced to the beans from the top of the vessel to avoid
drying out and to avoid loss of microorganisms. The beans are
placed into the vessel in a number of mono-layers on the perforated
trays to allow air for the bacteria. Exudate collection and/or
reintroduction is optional and depends on the degree of exudate
development.
[0041] Alternatively, the incubation of the vanilla beans with B.
subtilis can be performed in closed or vented bags, for example,
without limitation, plastic bags, or in a box, room or other
contained environment (for example on shelves), or even in an open
system, for example, outside exposed to ambient air, provided the
moisture content of the air is appropriately high (about 55%
humidity or higher).
[0042] The incubation temperature is from about 40.degree. C. to
about 65.degree. C., for example, without limitation, from about
45.degree. to about 60.degree. C., or from about 50.degree. C. to
about 60.degree. C.
[0043] Depending on the container used for incubation, and
depending on whether stirring is used, there may be a temperature
gradient in the container and care should be taken to ensure not
only a suitable average temperature but also that the maximum
temperature is not significantly exceeded for too long, in order to
avoid sporulation and ensure sufficient growth or at least survival
rates of B. subtilis.
[0044] The duration of incubation of the vanilla beans with B.
subtilis will depend on various parameters including temperature,
pH, etc., and whether a pre-culture is used or B. subtilis is
directly added from a stock culture (the latter requires more
time). Incubation should be stopped once the optimal vanillin and
vanilla flavour compound yield is reached. The incubation time
should be about 24 hours to about 120 hours. Usually 2 to 4 days
are sufficient. Longer incubation, especially at the higher
temperatures, will start to degrade vanillin and other vanilla
flavour compounds, longer incubation especially at the lower end of
the temperature range and especially when the beans are stationary
will promote the growth of molds and other undesirable
microorganisms that can spoil the product and cause off-notes.
Shorter incubation will not allow a complete degradation of
glucosides (glucovanillin and other glucose-bound vanilla flavor
compounds) and the product will not have a fully developed
well-balanced flavor profile.
[0045] After B. subtilis incubation, the vanilla beans may be
dried, or a vanilla extract may be directly formed.
[0046] Optional Drying Prior to Extraction:
[0047] Any suitable method may be used to dry the beans to a low
moisture content of about 30% to about 20% at temperature from
about 20.degree. C. up to about 60.degree. C. Suitable methods
include, without limitation, vented drying out in the sun or in a
well vented room, and drying in a vented oven.
[0048] To further develop the non-vanillin/non-glucose-precursor
vanilla flavour compounds part of the aroma including the part due
to Maillard reactions, slow drying will usually give better
results. A suitable drying schedule is to dry 2-5 days to about 30%
moisture, then for 3 to 9 months to about 20 to about 25% moisture
(storage in vented sacks in a well vented room for the latter
step).
[0049] To form a vanilla extract, a solvent may be added to the
incubation container, for example, without limitation, a tank, to
form a vanilla extract. Suitable containers include, without
limitation, a conical extractor/stirred tank, and a box extractor
wherein the beans are stationary.
[0050] After extraction, the resulting extract can be concentrated
or diluted as desired. The extraction temperature should not exceed
a maximum of about 45.degree. C. to avoid any partial degradation
of vanillin or other vanilla flavor compounds.
[0051] A vanilla extract consists substantially of extractive
matter of vanilla beans from vanilla plants and optionally a
solvent. For example, certain sugars may be added in some
countries. Solvents include, without limitation, alcohol/ethanol,
and water. Solvents that are not food-grade have to be removed or
sufficiently reduced in concentration to form the end product sold
to the consumer according to nationally differing regulatory
requirements.
[0052] Optionally, food-grade solvent may be added to adjust the
concentration or so-called fold of the vanilla extract end product
as desired.
[0053] Any solvent able to bring vanillin and/or additional vanilla
flavors into solution can be used. Useful solvents include water
and alcohols, including without limitation organic alcohols.
Organic alcohols include, without limitation, alkanols having up to
4 carbon atoms, for example, ethanol, isopropanol, propanol, low
molecular weight glycols and polyols including, without limitation,
propylene glycol, butylene glycol, or glycerin, or mixtures
thereof.
[0054] Suitable solvents include, without limitation, aqueous
alcohol solvents containing at least one alcohol miscible with
water in the proportions employed. Suitable aqueous alcohol solvent
includes, without limitation, mixtures of ethanol and water, for
example, ethanol/water (50:50).
[0055] If a food-grade solvent is used it does not need to be
removed afterwards, which is more efficient particularly in the
final process steps. Food-grade solvents include, without
limitation, ethanol, water, mixtures of ethanol and water,
ethanol/water (50:50), and mixtures thereof. Another alternative
may be supercritical fluid extraction.
[0056] Solvents that are not food-grade, or not food-grade in all
legislations or in all concentrations for all end products, for
example, without limitation, hexane, ethylmethylketone, methyl
acetate, dichloromethane, fusel oil, or mixtures thereof, have to
be either completely removed or at least partially removed (to a
specific low concentration that usually varies according to the
national food regulations) to form the end product sold to the
consumer. The term "fusel oil" designates a distilled concentrated
liquid obtained in small amounts as a by-product of alcoholic
fermentation and distillation that consists of a mixture chiefly of
alcohols, such as isopentyl alcohol, amyl alcohol, isobutyl
alcohol, isopropyl alcohol and propyl alcohol.
[0057] Optionally, the resulting vanilla extract can be
concentrated by at least partly removing the solvent.
[0058] Optionally, any residual glucovanillin (and other precursor
vanilla flavor compounds bound to glucose) in the resulting product
or extract may be converted to vanillin (or the relevant
non-glucose-bound vanilla flavor compound) by adding
beta-glucosidase (commercially available from various sources
including Biocatalysts Limited, Cardiff, UK) or a mixture
containing a beta-glucosidase enzyme and optionally additional
enzymes. The amount of beta-glucosidase enzyme depends on the
incubation parameters including duration, temperature, amount and
concentration of substrate and of solvent. Too much enzyme will not
have an additional benefit and may introduce off-tastes. If a
mixture of enzymes containing 5 units per g of beta-glucosidase is
used, then an amount of enzyme 0.005 to 0.2 times the vanilla bean
charge weight is usually sufficient, for example, without
limitation, 0.02 to 0.1, or 0.035 to 0.15 times.
[0059] A suitable solvent to use in the enzyme incubation includes
water and mixtures of water with another solvent compatible with
the enzyme. For example, ethanol should be less than 25% for
optimal enzymatic reaction.
[0060] The enzyme is incubated within a suitable temperature range,
for example, 20.degree. C. to 80.degree. C. or 40.degree. C. to
60.degree. C., for example, without limitation, near its
temperature optimum at about 55.degree. C. to 60.degree. C., and
held at this temperature for a sufficient time to convert
glucovanillin to the desired vanillin yield. Continuous stirring
will ensure a constant temperature and concentrations. Smaller
volumes may not require stirring.
[0061] The incubation time will depend on the amount and
concentration of the enzyme and substrate, solvent present,
temperature chosen, for example, without limitation, 1 hour to 2
days or longer. Usually, 24 hours are sufficient.
[0062] Preferably, the mixture should be stirred during the
enzymatic reaction to ensure sufficient mixing, constant
temperature throughout the reaction vessel, and access of enzyme to
substrate.
[0063] After the enzymatic incubation, optionally, the vanilla
extract may be concentrated and/or a food-grade solvent may be
added as described above.
[0064] The vanilla beans and their extracts described herein
provide a fully developed developed and well-balanced flavor
profile comprising the major vanilla flavor compounds. Major
vanilla flavor compounds include, without limitation, phenolic
compounds, furan compounds, fatty acid compounds, compounds formed
by reaction with ethanol, and acetaldehyde diethyl acetal.
[0065] Phenolic vanilla flavor compounds include, without
limitation, acetovanillone alpha-ethoxy-p-cresol, benzoic acid,
guaiacol, 4-methylguaiacol, p-hydroxybenzaldehyde, methylparaben,
methyl vanillate, 2-methoxy-4-vinylphenol 5-methoxyvanillin,
phenol, Vanillin, vanillic acid, vanillyl alcohol, vanillyl ethyl
ether, and p-vinylphenol.
[0066] These phenolic vanilla flavor compounds, in particular
guaiacol, should be present in a low concentration so that they do
not dominate, resulting in an unbalanced flavor.
[0067] Furan vanilla flavor compounds include, without limitation,
2-furfural, 2-furfurol 5-(hydroxymethyl)-2-furfural,
5-methyl-2-furfural, 2-hydroxyfuraneol, gamma-butyrolactone
(dihydro 2(3H)-furanone.
[0068] Fatty acid vanilla flavor compounds include, without
limitation, linoleic acid, and palmitic acid.
[0069] Vanilla flavor compounds that are formed by the reaction
with ethanol include, without limitation, etyl acetate, ethyl
glycolyte, ethyl lactate, ethyl linoleate, ethyl pyrovate, ethyl
levulinate, and diethylsuccinate.
EXAMPLES
[0070] Unless otherwise indicated, percentages are given as wt/wt
and all ingredients and solvents are food grade. Vanilla beans were
from Vanilla planifolia plants. Vanilla extracts below use the US
"fold" unit, e.g. a single-fold vanilla extract is one which
contains the extractive matter of one unit of vanilla beans (13.35
oz vanilla pods with a maximum moisture content of 25%) in one US
gallon (35% alcohol) of product. Thus, a ten-fold vanilla extract
is one which contains the extractive matter of ten units of vanilla
beans per US gallon of finished product according to the FDA, Title
21.
Example 1a
[0071] Fermentation of Green Vanilla Beans with Bacillus
subtilis
[0072] Reduction of Residual Microbes:
[0073] Green beans were exposed to hot water (about 85.degree. C.)
for about 1 to 4 min.
[0074] The green vanilla beans were fresh green vanilla bean with a
percentage of moisture that is usually about 85% (wt/wt), or (see
example 1b) dried green vanilla beans with moisture of less than
10% (as low as about 5%, which corresponds to completely dried
beans--the residual 5% water cannot be removed by conventional sun
or oven drying). The moisture percentage was determined using an
infrared oven (Moisture Analyzer, Computrac, Model Max-1000, by
Arizona Instruments) and weighing the beans before and after
drying.
[0075] B. subtilis Subtilis in 0.9% NaCl:
[0076] One tube of about 1 g of a lyophilized culture of a B.
subtilis subtilis strain NRRL B-50118, isolated from vanilla beans
and deposited Mar. 20, 2008 with the ARS Patent Culture Collection,
was dissolved in 20 ml sterile nutrient broth (Bacto.TM. nutrient
broth dehydrated for cultivation of non fastidious microorganisms,
Difco, Laboratories Detroit, Mich., USA, 8 g/l of distilled water).
The culture was incubated at 37.degree. C. at 200 rpm for 2 to 3
days until spores developed (spores are determined optically with a
microscope). 500 ml of nutrient broth was inoculated with the whole
of this culture, the resulting culture again was incubated at
37.degree. C. for further 2 to 3 days until spores developed. The
cells were precipitated by centrifuge and the precipitate was
redissolved in 5 liters of sterile 0.9% NaCl.
[0077] 12 kg of green vanilla beans were cut in to 2 cm pieces and
incubated for 1 hour at room temperature in 5 liters of B. subtilis
subtilis in 0.9% NaCl prepared as described above. These B.
subtilis subtilis exposed beans were filled into a curing tank
vessel at 2 kg beans/layer (to give nearly a monolayer) and 6
layers deep on perforated metal plates. The curing tank vessel
(also known as sweating tank) was a custom-made large-scale vessel
as described by McCormick 1973 in U.S. Pat. No. 3,663,238. The B.
subtilis exposed beans were cured at an average temperature of
about 50.degree. C. for 72 hours, with a maximum temperature of
60.degree. C., no exudate was collected.
[0078] The curing tank vessel was emptied and the beans were left
to dry slowly. The vanilla beans were dried with warm air at about
50.degree. C. until 60% moisture was reached (for about 12 hours),
then to 38% moisture at 40.degree. C. (for about 5 h), and at
ambient temperature of about 20 C until 30% moisture (for about
10-20 hours).
[0079] The resulting vanilla beans with moisture contents of 25 to
30% (wt/wt) were packed in plastic bags and stored at room
temperature avoiding exposure to sunlight for about 3 months.
[0080] 1.59 kg (or 3.5 lbs) of ground vanilla beans were extracted
and concentrated to give 1 times the vanilla bean charge weight of
the resulting extract. Vanilla beans were extracted five times with
50/50 (w/w) tap water and ethanol 2.27 kg or 5 lbs of solvent per
extraction. Ethanol was added to adjust to about 40% (v/v) ethanol
(about 0.3 times the vanilla bean charge weight) to achieve a final
weight of the resulting 10 fold vanilla extract equal to 1 times
the vanilla bean charge weight.
Example 1b
[0081] Fermentation of Dried Rehydrated Green Vanilla Beans with
Bacillus subtilis
[0082] Preparation of Dried Green Vanilla Beans:
[0083] Green fully ripe vanilla beans with a high concentration
(about 6% based on the dried weight of beans; usually green beans
have about 16% dry substance, and after drying, about 90% dry
substance) of glucovanillin determined by visual inspection (green
bean with yellow tip) were used. Drying was performed by cutting
the beans into pieces of about 2.5 cm length, and then incubating
at about 85.degree. C. (80.degree. C. to 90.degree. C.) in an oven
until a moisture concentration of less than 10% was reached. The
moisture percentage was determined using an infrared oven (Moisture
Analyzer, Computrac, Model Max-1000, by Arizona Instruments) and
weighing the beans before and after drying. The drying procedure
takes from about 10 to 20 hours. Without wishing to be bound by
theory, this may contribute to reduce microorganisms to an
acceptable level so that the formed vanillin is not significantly
degraded during fermentation.
[0084] 0.400 kg of the dried and ground beans were re-moisturized
with 2.0 liters of B. subtilis subtilis in 0.9% NaCl prepared as
described in example 1a in a stirred tank.
[0085] The resulting mixture was stirred for 96 hours at 50.degree.
C. Ethanol was added to this mixture to form a single fold vanilla
extract and mixed for 12 hours at 50.degree. C. then cooled to
25.degree. C. A single fold vanilla extract equal to 0.1 times the
vanilla bean charge weight was prepared.
Example 2 B. subtilis Strain--16 S rRNA Based Characterization,
Identification as B. subtilis Subtilis
[0086] B. subtilis subtilis strain NRRL B-50118, isolated from
Vanilla planifolia vanilla beans cultivated in Madagascar and
deposited Mar. 20, 2008 with the ARS Patent Culture Collection, was
identified by 16S rRNA sequencing over 500 by as follows.
[0087] The 16S rRNA gene was PCR amplified from genomic DNA
isolated from pure bacterial colonies. Primers used for the
amplification correspond to E. coli positions 005 and 531 (>500
by package). Amplification products were purified from excess
primers and dNTP's using Microcon 100 (Millipore) molecular weight
cut-off membranes and checked for quality and quantity by running a
portion of the products on an agarose gel.
[0088] Cycle sequencing of the 16S rRNA amplification products was
carried out using AmpliTaq FS DNA polymerase and dRhodamin dye
terminators. Excess dye-labeled terminators were removed from the
sequencing reactions using a Sephadex G-50 spin column. The
products were collected by centrifugation, dried under vacuum and
frozen at -20.degree. C. until ready to load. Samples were
suspended in a solution of formamide/blue dextran/EDTA and
denatured prior to loading. The samples were electrophoresed on an
ABI Prism 377 DNA sequencer. Data was analyzed using Applied
Biosystems DNA editing and assembly software and sequence
comparisons were obtained using the Microseq software.
[0089] The analyzed sequence showed a 0% mismatch (or 100% match)
over the analyzed >500 by to B. subtilis subtilis.
Example 3
[0090] Sensory Evaluation
[0091] 0.15 ml of the 10 fold of example 1a and 1 ml of the single
fold vanilla extracts of example 1b and as control commercially
available 10 fold and single fold extract from traditionally cured
vanilla beans (subjected to a standard curing process over several
months) were added to 100 ml of sweetened milk (5% sucrose) and
compared in a sensory evaluation by trained panelists.
[0092] Trained panelists compared the samples and the control.
[0093] All panelists noted a very clean well-balanced vanilla
profile without off-notes and a very strong vanillin note in sample
1a, which was much stronger than in the standard 10 fold vanilla
extract. The impact of vanillin was lower in sample 1b but the
panelists found the sample sweeter.
[0094] Panelists found all samples had a well-developed
well-balanced vanilla aroma.
Example 4
[0095] Consistent Flavor Profile
[0096] 5 batches were produced as described in example 1a, with the
same amount and concentration of B. subtilis, but with slight
variations in incubation temperature (40.degree. C. to 65.degree.
C.) and length, or washing steps (heat or rinsing with 70%
alcohol).
[0097] The resulting vanilla extracts were tasted by 5 trained
panelists.
[0098] Samples are evaluated based on a 1-10 preference scale, with
a control reference of high quality bourbon vanilla bean extract
(traditionally cured) with a vanillin concentration of at least
1.5%, good vanilla flavor and no detectable off-notes (8).
[0099] Vanilla extracts from commercial sources either
traditionally cured can range from 1 (least preferred, no
fullbodied vanilla flavor, unbalanced flavor profile, presence of
green, beany, fatty acid, moldy, or smoky notes) to 10 (perfectly
balanced vanilla flavor with low concentration of phenolic/guaiacol
off-notes, and no presence of any other off-notes). A good quality
commercially available vanilla extract usually ranges from 5 to 8.
Vanilla extracts formed not through traditional curing but through
various alternative processes perform significantly lower and need
to be mixed with better quality extracts for commercial sales.
[0100] Even though the batches varied slightly in temperature etc.,
all 5 batches fell within a narrow range at the higher end of the
1-10 scale (8-10), demonstrating a consistently high vanilla flavor
quality.
Example 5
[0101] Effect of Lack of Drying/Temperature and B. subtilis
Concentration
[0102] A vanilla bean extract was prepared as described in example
1a. It was compared to an extract formed from non-dried beans
naturally cured at 20 to 25 C (ambient temperature) on the vine and
no added B. subtilis apart from the naturally occurring bacterial
population. The sensory evaluation was performed as described in
example 4.
[0103] The extract of example 1 scored at 9, the naturally cured
beans at 1 (even though they had a high vanillin concentration of
3%).
Example 6
[0104] Conversion of Vanilla Flavor Precursor Compounds to Vanilla
Flavour by B. subtilis
[0105] Gluco-vanillin was added to a culture of B. subtilis
subtilis strain NRRL B-50118, isolated from vanilla beans and
deposited Mar. 20, 2008 with the ARS Patent Culture Collection. The
B. subtilis culture was prepared in nutrient broth (Bacto.TM.
nutrient broth dehydrated for cultivation of non fastidious
microorganisms, Difco, Laboratories Detroit, Mich., USA, 8 g/l of
distilled water) and grown to an OD600 of 0.6-2.0 prior to the
addition of gluco-vanillin.
[0106] After addition of glucovanillin, the B. subtilis culture was
incubated for 120 h at a temperature of 37.degree. C.
[0107] Samples were taken as indicated in the table below to detect
vanillin and various intermediates of the conversion of
glucovanillin to vanillin.
[0108] The results are indicated in the table below.
TABLE-US-00001 1 h 2 h 5 h 7 h 9 h 26 h 48 h 120 h Compound [ppm]
[ppm] [ppm] [ppm] [ppm] [ppm] [ppm] [ppm] gluco-vanillin 3300 3200
3000 2900 1300 100 0 0 guaiacol 1.35 1.90 6.48 14.3 306 139.6 207
295 benzoic acid 0 0 0 0 20.2 20.9 27.3 19.0 4-methyl- 0.23 0.10
0.07 0.07 2.97 10.2 13.3 25.0 guaiacol vanillylalcohol 55.3 18.8
20.2 35.4 31.3 107.2 192.9 144.6 vanillic acid 0 142 141 163 200
200 300 800 vanillin 50 80 200 210 40 80 70 30
[0109] Gluco-vanillin was completely digested to vanillin within 26
hrs. The concentration of vanillin increased to 210 ppm at 7 hours,
and then decreased again. The concentration of 4-methylguaiacol,
benzoic acid, vanillylalcohol, and vanillic acid increase
relatively constantly over the measuring time, except for some
minor fluctuations, especially for the lower or higher
concentrations (for example guaiacol and vanillyl alcohol).
[0110] In a second experiment, the same B. subtilis strain was
incubated as described above with the glucoside of
p-glucobenzaldehyde.
[0111] Phenol can be formed from p-glucobenzaldehyde (p-GBA)
enzymatically, for example by beta-glucosidase which forms
p-hydroxybenzaldehyde (p-HBA), and then
p-hydroxybenzaldehydedehydrogenase which forms p-hydroxybenzoic
acid (p-HBAc), and in the last step p-hydroxybenzoatedecarboxylase
which forms phenol.
[0112] The fermentation broth was analyzed in regular intervals by
High Performance Liquid Chromatography/HPLC (non-volatiles) or Gas
Chromatography/GC (volatiles, phenol) as indicated in the table
below.
[0113] The results are indicated in the table below.
TABLE-US-00002 1 d 6 d 9 d 14 d 19 d [ppm] [ppm] [ppm] [ppm] [ppm]
p-GBA 0 (0.5) 0 0 0 (HPLC) p-HBA 239 204 176 46 2 (HPLC) p-HBAc 27
83 115 244 285 (HPLC) Phenol 0.59 8.58 8.35 23.15 33.9 (GC)
[0114] The results show that over the time the concentration of
phenol increases, while p-glucobenzaldehyde decreases rapidly to 0
within 24 hours (except for one measuring point). At the same time
the intermediates (p-hydroxybenzaldehyde and p-hydroxybenzoic acid)
have formed, and with continued formation of phenol, p-HBA
decreases again. The measurements of phenol analyzed by GC are not
directly comparable but show that phenol is formed and increases
over time, whereas its precursor and intermediates decrease.
[0115] While the processes and products have been described above
in connection with certain illustrative embodiments, it is to be
understood that other similar embodiments may be used or
modifications and additions may be made to the described
embodiments for performing the same function(s). Further, all
embodiments disclosed are not necessarily in the alternative, as
various embodiments may be combined to provide the desired
characteristics. Variations can be made by one having ordinary
skill in the art without departing from the spirit and scope of the
disclosure. Therefore, the processes and products should not be
limited to any single embodiment, but rather construed in breadth
and scope in accordance with the recitation of the attached
claims.
* * * * *