Note: Descriptions are shown in the official language in which they were submitted.
- 1 328770
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DESCRIPTION
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NATURAL BUTTERY COFFEE FLAVOR BY FERMENTATION
FIELD OF THE INVENTION
The present invention is related to the field of the
flavoring of food products, specifically the addition of a
natural flavor formed by fermentation. The invention involves
the production of a natural buttery and/or winey flavor of which
diacetyl and acetoin are the major flavor constituents from the
fermentation of a coffee substrate by a strain of lactic acid
producing bacteria or yeast capable of producing diacetyl. The
fermented coffee material which has a buttery, winey flavor, may
be added directly to the green coffee to be processed therewith,
-or the flavor may be distilled or concentrated and added to a
soluble coffee product or a roast and ground coffee product.
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1 ~a~KGROUN~ ART
Food technologists have over the years strived to
3 develop synthetic or natural flavors which would either
impart a coffee-like flavor to an imitation beverage or a
;; 5 flavor which would augment a particular flavor note in a
natural coffee product in which the flavor note was
7 deficient. A compound in coffee which imparts a butter
flavor and aroma is the compound diacetyl (2,
9 3-butanedione). This compound is a yellow liguid having
an estremely potent butter aroma. Diacetyl can be formed
11 either by chemical synthssis or by bacterial
fermentation. Diacetyl as a flavorant is particularly
13 important in dairy products such as sour cream, and
cultured butter and butter milk. Also it is important in
15 such non-dairy products as butterscotch, honey, coffee,
maple syrup, white bread, summer sausage, and margarine.
17 United States Patent No. 4,304,862, issued to Troller
entitled ~Method for Increasing the Diacetyl Production
19 of a Diacetyl-Producing Bacteria~ teaches a method
wherein the increased diacetyl production i~ attributable
21 to a set of specific conditions including a nutrient
medium having a pH of from about 4.5 to 7, incorporation
23 of a humectant to lower the aw value of the nutrient
~ medium to about 0.95 to 0.99 and a tem~erature for
- 25 incubation of from about 28 to 37C. The patent also
teache~ incorporating a metabolizable amount of a
27 diacetyl precur~or which is citric acid or an acceptable
Jalt thereof. An article written by Collins in The
; 29 Journal of Dairy Science entitled ~Bio8ynthesis of Flavor
Compounds by Microorganisms~ teaches that the enhancement
31 of diacetyl production requires a strain of bacteria or
yeast able to produce diacetyl, and the control of the
33 environment such as to achieve good growth of the
selected organism. Specifically the environment includes
35 a pH below about 5.5 and an osygen containing environment.
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1 While diacetyl has been known aS an important flavor
ingredient of dairy and coffee products, fermentation
3 produced by bacteria or yeast on a coffee substrate to
produce a natural diacetyl has not heretofore been
5 demonstrated. It is an object of the present invention
to produce diacetyl by microbial fermentation of pure
7 coffee materials to produce a buttery flavor directly
from a coffee substrate.
DISCLOSURE OF THE INVENTION
11 The present invention i~ a method for producing a
natural buttery and/or winey flavor of which diacetyl and
13 acetoin are the major flavor constituents from a coffee
substrate. The process involves orming a nutrient media
15 containing water and soluble coffe~ solids as the sole
nutrient component, that is the component which promotes
17 the growth and development a~ well as acts a~ the raw
materials from which new protoplasm can be synthesized,
19 the soluble solids concentration being at least 0.5%. A
microorgansim which is either a strain of lactic acid
21 producing bacteria or yeast capable of producing
diacetyl, preferably a lactic acid producing bacteria is
23 added the nutrient media and the combination
microorganism and nutrient media is gently mised in order
25 to brinq into the nutrient media a certain amount of air
and si~ultaneously placed into an environment of a pH of
27 from 4.0 to 7.0, a temperature of from 16 to 37C, for a
period of time effective to produce diacetyl and aeration
29 conditions wherein the coffee substrate composition
contains sufficient osygen to allow for diacetyl
31 production. The aforementioned process will produce an
amount of diacetyl up to 2000 parts per million (ppm) and
33 variable amounts of acetoin depending upon the time of
fermentation.
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1 As a result of the process of the present invention,
a buttery and/or winey flavor is produced naturally from
3 pure coffee material and as a result if the flavorant is
added to a coffee beverage the combination can be termed
5 ~all coffee~. Addition of the natural flavoring produced
according to the present invention will provide a
7 smoother, ~buttery and more mellow brew.
9 BEST MODE FOR CARRYING OUT THE INVENTION
The present invention resides in a process for
11 producing a natural buttery and/or winey flavor
consisting primarily of diacetyl and acetoin which are
13 produced from a coffee substrate. The microorganism
which is utilized to ferment or ~bioconvert~ the coffee
15 substrate to produce the natural flavor include any
strain of lactic acid producing bacteria or yeast capable
17 of producing diacetyl, preferably a lactic acid producing
bacteria. Suitable lactic acid producing bacteria
l9 include the families Lactobacillaceae, Streptococcaceae,
and also certain species of Micrococcus which generate
21 lactic acid. Preferred bacteria for carrying out the
` present invention include pure cultures or mistures of
f23 strains of the following species: ~C~ L l~
StreDtococcu~ diacet~lactis, Leuconostoe citrovorum,
25 Streptococcu~ cremoris, Stre~tococcus faecali~, and
~actobacillu~ ~ylosus, for the reason that both growth
27 and diacotyl production occurs on coffee substances.
Suitable coffee substrates employed in the present
29 invention as part of or all of the all-cofee nutrient
media include soluble solids from green e~tract (aqueous
31 green coffee ~olids) ground qreen coffee beans, coffee by
products (pulp, coffee husks and mucilage), as well as
33 hydrolyzed spent grounds, roast and ground coffee and
brown e~tract. The preferred substrate concentration
35 will vary from 0.75% to 25% soluble solids
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~ ~ 5 ~ 1 32~770
1 concentration. Too high a concentration of coffee solids
will create an osmotic problem causinq cells to loose
3 water to the nutrient media. Soluble coffee solids as
defined in the present invention shall include
5 e~tractable coffee solids. Of the a~ove listed coffee
substrates, green estract containing the above-identified
7 concentration of soluble green coffee solid~ is the
preferred coffee substrate. In a preferred mode, the
9 nutrient media is made up esclusively of soluble coffee
solids alone or in combination with pyruvate.
11 The microorganisms which have been added to the
nutrient media containing soluble cofee solids are
13 placed into an environment in which the conditions of pH,
temperature, time and aeration conditions must be kept
15 within certain defined limit~. The pH of the bacteria or
yeast and coffee substrate combination must fall within
17 the range of 4.0 to 7Ø The temperature should be
between 16 to 37C, preferably from 27 to 32C. The
19 bacteria or yeast should ferment in contact with the
coffee substrate composition for a period of time
21 effective to produce diacetyl, preferably from 8 to 96
hours.
23 Aeration is an important part of the bioconversion in
that osygen Qtimulates diacetyl production. Literature
~l 25 states that osygen plays a role in diacetyl production by
; reducing the amount of pyruvic acid which must be
27 converted to lactic acid for NADH. Osygen also
; influence8 the formation of diacetyl by influencing the
29 formation of acetyl CoA. As is apparent to those
knowledgable in the art of biochemistry, the osidation of
31 1 mole of glucose produces 2 mole~ of pyruvic acid, and
2 moles of NADI are reduced (Glycolysis). The supply of
33 NAD+ is limited, and the reduced NAD+ must be reosidized
so that the osidation of glucose may continue. Pyruvate,
35 produced via glycolysis, is used as a proton acceptor
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;1 (electron donor) and is reduced to lactic acid. In the
presence of osygen, NADH o~idase is stimulated to produce
3 NAD~, and this lowers the metabolic demand of its
production via pyruvate reduction.
The biosynthesis of diacetyl also requires acetyl
CoA. Lipoic a~id is involved in the formation of acetyl
7 CoA from hydrosyethyl-TPP and Coenzyme A (pyruvate
dehydrogenase comples). In this mechanism, the lipoic
9 acid is reduced; NAD+ is necessary for reo~idation of
lipoic acid and in turn is itself reduced. NADH o~idase,
11 whose enzyme actvity is stimulated by osygen (aeration),
is invol~ed in the reosidation of NAD~.
; 13 An optional minor ingredient which may be added to
the nutrient media is a diacetyl precursor or enhancer,
15 preferably pyruvic acid or bacteriologically acceptable
salts of pyruvic acid which is added at a lsvel of from
17 about 0.1% to 1.0%, preferably from 0.5% to 0.8~ by
,weight of the nutrient media. The pyruvate will function
;,19 to increase the amount of diacetyl produced by two to ten
fold. The pyruvic acid which is added may be either
~21 naturally produced or sythetically produced. Most
Apreferably the pyruvic acid is a natural, coffee-derived
23 ingredient thereby allowing the diacetyl so produced to
be termed a natural additive produced from an ~all
25 coffee~ source.
Th- conditions to which the bacteria are subjected
27 are not conditions optimal to its continued growth and
reproduct~on. At the conditions specified above, the
29 organism i5 not metabolizing efficiently. The bacteria
will, however, thrive on nutrient media anaerobically at
31 a temperature of appro~imately 37C and a pH of 6.5.
A continuous or semi-continuous process for producing
33 diacetyl from a coffee substrate may also be accomplished
by the present invention. Thi~ type of process would
35 necessitate the use of an immobilized bacteria or high
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1 substrate concentration. One of several different
methods can be utilized to accomplish the immsbilization
3 of one or more of the diacetyl producing strains of
lactic acid bateria. These include encapsulation,
S attachment to a solid support, isolation utilizing a
membrane with a pore size smaller than the bacteria or
7 other means readily apparent to those skilled in the
art. For further discussion on the use of immobilized
9 cells, attention is directed to the article entitled
- ~Application of Microbial Enzymes for Production of
11 Food-Related Products~ by Weetal and Zelko.
In such a continuous or semi-continous system it may
13 be desirable to subject the immobilized cells of the
bacteria to conditions optimal for diacetyl production
15 and then alternatively to a differsnt set of conditions
which are optimal for bacterial growth ~approsimately
17 37C, anaerobic environment and pH of 6.5). During
optimal bacterial growth it may be desirable to utilize
19 conventional nutrient media as the growth substrate since
it will provide for faster bacterial growth.
21 An alternative method for continuous or
semicontinuous production of diacetyl is to u8e the pH,
23 temperature and aerations condition~ described
previou~ly, but also proYide a regulated flow of soluble
25 green coffee solids at a 7.25% concentration. The system
whon oporated under these parameter~ produces both
21 diacetyl and ~low growth of the bacteria. The flow is
matched to the ~low growth rate of the bacteria under the
29 above conditions. Although less efficient in producing
diacotyl from soluble solid on a weight per weight basis,
31 this system has overall process advantages if the
fermented solids can be returned to the coffee product.
33 After the effective period of time, an amount of
diacetyl i3 produced according to tho process of up to
35 2000 parts per million where pyruvate is added and at
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1 levels approsimatelY 200 parts per million without the
additional pyruvate or other diacetyl precursor. An
3 amount of acetoin is also produced at levels which may bs
smaller or larger than the amount of diacetyl depending
5 upon the length of fermentation time. The solution
containing the diacetyl, acetoin, coffee substrate and
7 bacteria could be added as is to green coffee, roast and
ground co~fee, or to brown estract. Preferably, however,
g the diacetyl and/or acetoin which is produced is isolated
from the other compounds present by distillation
11 according to known means. The diacetyl will bc
incorporated into a cofee product or an imitation
13 coffee-type product at a level of from 0.1 to 1.5 parts
per million on an as-consumed basis.
The utilization of this invention has application to
soluble coffee processing. After the diacetyl i~
17 recovered and concentrated, it may be added at any
convenient step in the soluble coffee process such as
19 plating the dry soluble coffee or coffee substitute with
a desired dilution of the flavor agent in an acceptable
21 solution followed by drying. In certain instances the
diacetyl may be added either in solution with the coffee
23 ~ubstrata and bacteria or it may be i~olated and then
added directly to a concentrated coffee estract and the
25 misture spray dried or freeze dried into a soluble coffee
product which contains the diacetyl as an intrinsic part
27 thereof. The diacetyl is added to a soluble coffee
product at a level of from 10 to 150 ppm diacetyl on a
29 dry weight ba~is.
The diacetyl may also be added to roast and ground
31 coffee to augment a buttery flavor note. The diacetyl
can be added to roast and ground coffee at a level of
33 from 50 to 400 ppm on a dry weiqht basis. The roast and
ground coffee containing the added diacetyl when brewed
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l will produce a coffee containing the aforementioned level
of diacetyl on an as-consumed basis.
3 The invention will be further described with
reference to the following e~amples which are given for
5 illustrative purposes only.
; 7 EXAMPLE 1
9 A natural buttery coffee flavor was produced
utilizing a whole green bean e~tract and separately from
ll a ground green bean e~tract.
Whole green beans, in a ratio of 20~ bean/80% water
13 (wt/wt) were eYtracted using an overhead ~tirer as
follows: beans were e~tracted for 2 hours at 60C after
15 which the temperature was increased to 77C (1 hour) and
held at 77C for 2 hours. The estract was espressed
17 through cheese cloth and concentrated to about 12~ solids
by rotoevaporation at 50C. The concentrated e~tract was
19 then autoclaved (~15 min e 120 psi). Prior to
innoculation with ~ s~i 393, pyruvic acid was added
21 (0.75% and the pH adjusted to 5.67. The misture was
innoculated (with appro~imately from 107 to 108 cells per
23 ml) viable c~lls of L._g~s~i and held at 28 to 29C.
Diacetyl levels of over 1000 ppm were obtained in
' 25 48 hour~.
Greon beans were ground in a Retsch mill. The
27 procedure used a~ove was followed with the addition that,
, after autoclaving, the e~tract wa~ centrifuged for
29 20 minute~ at 6000 rpm. Over 300 ppm diacetyl was
obtained without the addition of pyruvic acid under
31 similar conditions after 72 hours at 28C temperature.
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1 EXAMPLE 2
Diacetyl levels of 225 ppm were produced by
3 Lactobacillus Casei ATCC No. 393 bacteria after a 72 hour
batch fermentation in a 2-liter bioreactor operated under
5 aerobic conditions. An aeration rate of 0.1 volumes
air/volumes broth per minute (VVM) maintained nearly
7 saturated osygen conditions. The temparature was
maintained at 27C throughout the course of the run.
g Fermentation broth consisted of an all-coffee 10% soluble
solids estract (green soluble coffee solids) inoculated
ll with 107 cells/ml. Cell counts reached lO9 cells/ml
after the same period of time while the pH decrea8ed f rom
13 an initial value o 5.2 to 4.3.
An identical bioreactor maintained under strictly
15 anaerobic conditions by sparging nitrogen gas at a rate
of 0.1 VVM through the fermentation broth produced less
17 than 30 ppm diacetyl.
Despite the difference in diacetyl levels, cell
19 growth and pH a~ a function of time were identical to the
aerobic bioreactor thus indicating the importance of
21 osygen for diacetyl production.
23 ~XA~oeLE_l
A natural buttery coffee flavor wa~ produced in an
25 airtight 500 ml flask, utilizing a sterile 1% solution
containing 2.5 grams of soluble green coffee ~olids. The
27 solution wa~ then inoculated with 2.5 s 101 cells of
~a5~2b~sillius casei (ATCC No. 393) and placed in a
29 shaker to agitate slowly (60 rpm at room temperature
(23C)) at a pH of 5.4. After 50 hours, the culture
31 contained 60 ppm diacetyl. Even though th~ flask was
airtight, suficient o~yqen for diacetyl production was
33 present in the headspace because the volume of headspace
was large compared to the volume of inoculum and nutrient
35 media. Also samples were withdrawn two to three times a
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1 day allowing for some aeration. Greater aeration would
have increased the yield of diacetyl significantly.
3 The culture containing diacetyl was then removed from
the shaker and distilled at 100C. The distillate,
5 containing concentrated diacetyl (900 ppm) was then added
to a 1% solution of instant coffee at a 0.75 ppm level.
7 This addition resulted in a significant enhancement of
coffee flavor by producing a more ~buttery~ taste in the
9 coffee as judged by an e~pert panel. The concentrated
flavor adjunct was analyzed both organoleptically and
11 analytically via gas chromatography and found to contain
diacetyl as the major flavor compound.
13
Diacetyl was produced by incubating 2.5 grams of
brown estract solids which were dissolved in 250 ml. of
17 water with Streptococcus lacti~ subspecies diacetylactis
(ATCC No. 11007). The incubation misture contained
19 appro~imately 101 viable cells. These cells were placed
- in an airtight 500 ml. flask along with the
21 previously-mentioned solution of brown estract solids and
the misture wa~ agitated slowly in a shaker bath at
23 23C. The pH of the reaction misturs was 4.B. After a
period of 24 hours the misture contained 7 ppm diacetyl.
25 Even though the flask was airtight, sufficient osygen for
diacetyl production was present in the headspace because
27 the volume of headspace was large compared to the volume
of inoculum and nutrient media. Also samples were
29 withdrawn two to three times a day allowing for ~ome
aeration. Greater aeration would have increased the
, 31 yield of diacetyl significantly.
The mi~ture containing the diacetyl was filtered to
33 remove the bacteria and was than diluted ten fold with
fresh brown estract or dissolved instant coffee
35 containing 1% soluble solids. In either case the level
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1 of diacetyl in these coffee beverages as-consumed was
approsimately 0.7 ppm. The coffee bev~rage containing
3 the diacetyl possessed an enhanced ~buttery and winey~
flavor.
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- 7 Continuous production of diacetyl was attained for
209 hours by immobilizing cells of L. Casei (ATCC
9 No. 393) in a 180 ml stirred reator. The cells were
retained in the reator by an ultrafiltration membrane
11 ~Amicon Corp. PM-30) which retain substances with
molecular weights greatsr than 30,000 daltons. A
13 nitrogen pressure of 20 p~ig was used to maintain a flow
rate of 6.8 ml/hour of sterile 20% rich qreen coffee
15 estract throuqh the reactor. For continous production of
diacetyl at 30 ppm, 108 viable cell~ml were maintained
17 in the reactor, the pH was kept at 4.6-4.8 under nitrogen
atmosphere and the temperature at 23C. This very low
19 level of diacetyl was produced due to the lack of
osygen. Low levels of diacetyl may be produced under
21 anaerobic conditions but such conditions are
uneconomical. Higher levels of acetoin are produced
23 under anaerobic condition~.
On di~tillation of the filtrate, the distillate had
25 the flavor enhancing properties when added to ~oluble
coffee de~cribed previously in Esample 3.
27
EXAMPLE 6
; 29This esample demonstrates the production of diacetyl
by yeast fermentation of an all coffee substrate.
31 Saccharomvces uvarum (5.5 s 107 yea~t cells per ml.) was
-~ innoculated into a 1 litsr graduated cylinder containing
33 1,000 ml of green estract (approsimately 10~ soluble
solids) and allowed to ferment 3 day~ at room
35 temperature, pH 5.5, without agitation. After 3 days,
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1 the yeast cells were centrifuged out of the medium and
the spent medium was reinocculated with Candida boidinii
3 (1 s 1o8 yeast cells per ml.)and stirred at room
~ temperature, pH 5.25 at 160 rpm. Diacetyl was detectable
;~ 5 at 96 hours at a level of appro~imately 6 ppm.
7 EXAMPLE 7
This esample demonstrates the production of diacetyl
9 and acetoin utilizing coffee by products, specifically
coffee husks, as the source of soluble coffee solids. A
11 long neck culture flask containing fresh coffee husks
which wers ground and diluted (10% solids, ph 4.9) was
13 inoculated with 108 cells/ml L. Casei (ATCC No. 393) and
the combination was incubated at 27C and stirred at
15 200 rpm. After fermentation for a 72 hour period the
coffee medium was analyzed and was found to contain
17 327 ppm diacetyl and 245 ppm acetoin.
The above-identified conditions were repeated with
19 the esception that sodium pyruvate was addsd at a level
representing 0.75% by weight of the free acid based upon
21 the total substrate volume at the beginning of the
fermentation. After 48 hour~ 1013 ppm diacetyl and
` 23 919 ppm acetoin were produced. At 72 hour~ 693 ppm
diacetyl and 1129 ppm acetoin were produced.
EXAMPLE 8
27 A ~crewcap flask containing green coffee e~tract (10
301ids, pH 5.5) was inocculated with 108 cell~/ml
29 L. Ca~ei ~ATCC No. 393) and inculated at 29-C and stirred
at 200 rpm. After 48 hours sodium pyru~ate (0.75~ by
31 weight of free acid) was added and the fermentation
continued. Analysis of the coffee medium after 72 hours
- 33 of fermentation (24 hours ater pyru~ate addition)
yielded 909 ppm diacetyl and 2281 ppm acetoin. In
35 general, the longer the fermentation progresses after
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1 appro~imately 3 days, the higher the ratio of acetsin to
diacetyl in green coffee estract systems.
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