Note: Descriptions are shown in the official language in which they were submitted.
llZ~3357
1 ; Background of the Invention
Technical Field
. .
This invention xelates to a continuous process for
reducing the levels of certain nitrogen-containing compounds
S present in tobacco materials using microorganisms. Specific-
ally, the present invention provides a process for reducing the
levels of nitrates, nitrites and ammonium compounds via an
aerobic assimilatory metabolic pathway employing condi~ions
such that continuous, rather than batch, operation is possible.
Description of Prior Art
- , ; It is generally recognized that smoking products
! having lowered amounts of oxides of~ nitrogen present in smoke
, are desirable. Therefore, a number of methods have been
; developed to reduce the delivery of oxides of nitrogen by
smoking products. Among these techniques are various methods
wherein the nitrate content of the tobacco is altered. For
examFle, methods involving microbial treatment of tobacco to
accomplish s~ch nitrate reduction have been proposed.
; Specifically in Gaisch et al. Belgian Patent 886,445- 20 published August 14, 1978 and assigned to Fabriques de Tabac
Reunies S.A. a process for degrading nitrates and nitrites in
tobacco to nitrogen or ammonia compounds by means of micro-
organisms which would normally require oxygen, but are capable
of anaerobic denitration is decribed. Gaisch et al. German
~ Offenlegungsschrift 28 16427, filed April 15, 1978 and published
- November 9, 1978, describes a process for microbial degradation
. of nitrate, nitrite and other nitrogen containing compounds in
tobacco. Adcording to Gaisch et al., under nitrogen deficiency
or oxygen deficiency conditions, the microorganisms employed
obtain their nitrogen or oxygen requirements respectively from
.
., . ~ .
-2-
112~ 57
n_crate or nitrite degradation. The microorganisms which can
be used in these two processes may be selected from the genus
Aerobacter,~Pseudomonas, Micrococcus or Escherichia, with
Enterobacter aerogenes being specifically employed in the
examples.
European Patent Application 79 300 706.3 publ~shed
October 31, 1979, describes a process for microbial reduction
of nitrates in tobacco via a dissimilatory denitr~fication
pathway whereby nitrogen gas is the end product. The micro-
organism specifically suggested for use in the process is
Paracoccus denitrificans or Micrococcus denitrificans. Species
o~ the genera Pseudomonas, Alcaligenes, Bacillus and
Propionibacterium can also be employed.
Further U.S. Patent 3,845,774 to Tso et al. describes
tobacco treatmen~ methods referred to as homogenized leaf
curing wherein the tobacco is homogenized and incubated during
curing in order to regulate the composition of the final product.
Nitrate-nitrogen and total nitrogen are reduced somewhat;
however, the amount of reduction is not as significant as that
of the present process. Although Tso et al. allude to the fact
that tobacco modification can be accomplished by the use of
. .
additional techniques during homogenization and incubation,
such as enzyme ànd microbial action, no specific methods or
means for reducing nitrate-nitrogen are suggested.
ll Gravely et al., U.S. Patent 3,?47,608 relates to a
j¦ method for aerobic microbial digestion of pectin-bound plant
material, specifically tokacco materials. Although the invention
deals predominantly with methods for fibrilating tobacco
Il materials using pectolytic enzyme-producing microorganisms,
j,
11 Examples 1l and 13 disclose data related to the concom~tant
' ~! ' .
I -3-
' il . .. .
;~
li 1~2~3~7
i jlenitration of tobacco using the microorganism Erwinia carotovora,
' ATCC 495. This microorganism is unsuitable for use in the
present invention since pectolytic enzyme-producing micro-
' organisms, such as Erwinia carotovora, destroy the structural
S lintegrity of the tobacco.
W. O. Atkinson et al. reported a redu~tion in various
!tobacco leaf components, including nitrate-nitrogen~ by varying
. ~Ihomogenization and incubation techniques during curing.
~1 (Abstract of Proceedings of the University of Kentucky Tobacco
!land Health Research Institutet Lexington, Rentucky, Conference
Report 4, ~arch 1973, pages 829-33.)
¦ Denitration by means of microorganisms is also known
outside the tobacco arts. Representative examples are U.S.
Patents 3,709,364 to Savage, 3,829,377 to Hashimoto, 4,039,438
to Anderson, and 4,043,936 to Francis et al. which describe
denitrification of waste water using anaerobic bacteria to
¦reduce the nitrate to nitrogen gas. Members of the Thiobacillus,
¦Pseudomonas, Chromobacter, Bacillus and Clostridium genera are
. _ _
; lamong the mi~roorganisms which may be employed. In the
jHashimoto patent the use of pressuriæed systems to increase the
¦¦amount of methane available to the microorganisms and to
; l~acilitate liberation of the nitrogen gas by venting are sug-
I ; Iges~ed. The Anderson patent suggests conducting the process at
. j~bient or atmospheric pressure. In the Francis patent the
nitrogen gas passes through an exit out of the system. The
ISavage reference employs pressure to pass the effluent being
- !treated through the filter containing the microorganisms.
: I Microorganisms have also been used to modify other
Itobacco components. For example, U.S. Patents 4,037,609 and
io 1l4,038,993 to GeiB~ et al. disclose methods for xeducing the
l! '
11 ' . .
~, " ' " :
. !
4-
.
,
1 l~ icotine content of tobacco by microbial treatment using micro-
organisms obtained from tobacco, including Pseudomonas putida
and Cellulomonas sp. Aerobic fermentation techniques are
¦ employed wherein nicotine is degraded via microbial action to
¦ 3-succinoylpyridine. The latter microorganism is capable of
I reducing nitrate to nitrite and actively produces nitrogen gas.
¦I Similarly degradation of nicotine to 3-succinoylpyridine
~ by means of the same microorganisms is described in U.S. Patent
; ¦j 4,011,141 to Gravely et al. Lippman et al. U.S. Patent 2,000,855
o !¦ describes microbial denicotinization of tobacco by fermenting
.. . .
jl moist tobacco while adding acid to overcome the alkaline con-
i; dition produced by fermentation. Alternatively the patent
il suggests removal of volatile bases by supplying an air current
or employing suction. Fermentation was used to improve aroma
and mellowness in U.S. Patent 2,644,462 to Frankenburg and in
¦ U.S. Patent 4,135,521 to Malan et al.
; Further, U.S. Patent No. 2,149,179 relates to an
¦ accelerated aging method for tobacco wherein the aging is
¦ effected by means of fermentation with exclusion of oxygen
Il employing microorganisms capable of growing in the absence of
' oxygen. The microorganisms may be those which are bred on
, noble tobaccos or anaerobic yeasts. By means of the process,
!i fermentation times of only days~ rather than months are required.
; l¦ The purpose of the claimed fermentation process is to improve
the bouquet of the tobacco. Nicotine content in the tobacco is
¦l also rbduced. According to the patent, a prior process of
I! Suchsland, which used microorganisms to decompose complex
¦¦ organic substances in tobacco into simpler compounds, did not
¦ prove practical since the oxidation effected by oxygen during
I ~he ~ermentation was lgnored.
1 . ;
',.' ", I " . ' ' '
....... . .
~129;~S7
.
1 ~ We have now unexpectedly discovered that by employing
j,carefully controlled conditions, it is possible to effect
deni~ration via.an aerobic assimilatory metabolic pathway on a
continuous basis. Specifically it has been discovered that by
. ~~controlling the denitration conditions, it is possible to
coordinate the microorganisms' growth rate with the tobacco
extract treatment rate, whereby a denitration process is pro-
vided which i9 easily adapted to other continuous tobacco
treatment processes, can be employed on a continuous basis for
1~ lextended perioas with relatively little or no supervision and
- 'Ipërmits treatment of greater amounts of tobacco extract and
'i '
;Iresults in a higher production rate relative to batch processes.
. ~jThat is,.the present..process provides a method whereby nitrates,
~nitrites and ammonium compounds can be efficiently eliminated
1 .......... . . .
. . Ifrom tobacco via an-assimilatory metabolic process on a large,
technical scale under economical conditions, with a minimal
I! re:quirement of manpower or energy and minimal addition to or
,., .. il' ' '
j.transformations of the tobacco extract components,~other than
~¦such denitration.
: . 20 . il ~ Brief Description of the Drawings
'jl ' , .
FIGURE 1 is a flow diagram of a tobacco denitration
..sy~tem from the extraction of tobacco through the denitration
!.steps of the present invention.up to the reapplication of
.
.the denitrated extract to the extracted tobacco.
: Summary of the Invention
!,
: A continuous method for denitrating an aqueous
; tobacco extract which comprises contacting extract with a work
: mixture containing tobacco extract and microorganisms, which
: ~re capable of metabolic, aerobic assimilation of nitrogen-
containing compo~nds and which are in exponential growth phasa,
~ ' ", . . .
.
,
. ' -6-
, - .
/ :l
2~i7
1. 1, while maintaining pH, temperature and aeration at levels which¦ promote aerobic as~imilation, by adding the extract to the work
, . mixture at a dilution rate which does not exceed the growth
. . I i^ate of the microorganisms while addi.tionally adding phosphate
5~ ' ¦ and a carbon source to the work mixture, said extract, phosphate
,. ¦ 'and carbon source being sterile when added and being added in
., I amounts such that the overall addition thereof is 0.1-7.5 g
"' '!i ~itrate/l added, l.O,to 10 g PO43 /1 added and sufficient carbon
. Ij;source to provide at least 16.5 assimilative carbon atoms /NO3
' ~¦ molecule added, while withdrawing a portion of the work mixture
j àt a rate such that the volume of work mixture remains constant
.I and thereafter removing the microorganisms from the withdrawn
mixture., Prefer~ed microorganlsms for use in the present
.!1 process are Candida yeasts. Denitration with such yeasts may
~ ~I, be e,ffected continuously as above described employing a dilutio~
¦¦ and.withdrawal rate of O.l to 0.35 liter of addltives and
¦l extract per liter of work mixture per hour while maintaining a
Il pH of 3.5 to 7.2, a temperature of 25 to 37C and an aeration
I¦ rate of 0.8 to 2.5 liters air per liter work mixture per
;~ 20 ¦I minute. Enterobacter aerogenes may also be used in the present
.. . I ,process~ Denitration with such microorganisms may be effected
I .
!j on a continuous basis employing a dilution and withdrawal rate
of O.l to 0.25 liter of additives and extract per llter of
work mixture,per hour while maintaining a pH of 5..5-8, a
. l, temperature of 30-40C and an aeration rate of 1.0 to 3
¦ liters air per liter work mixture per minute.
. !l .
. ....... ,! Detailed Descri~tion of the Invention
, . , _ . _
,1 The present invention provides a met.hod whereby
,,' ¦~denitration.of tobacco extracts may be effected in a continuous ;
, 30 ', manner employing microorganisms capable of aerobic a5~imilation
,,' I .,
". i .' , '
., . I .
I . . 7
' ' :1
'7
of nitrate, sometimes referred to as nltrate ammonification.
It is possible to run a system employing the denitration method
of the invention for extended periods with little or no super-
vision.
Continuous assimilatory denitration is effected
according to the present invention by introducing material to
be treated into a fermentation vessel while withdrawing treated
extract from the fermentor at the same rate, such that the
overall volume of material in the fermentor, that is, the work
;10 mixture, remains constant. Moreover the rate of extract
introduction and withdrawal and the process conditions are such
that there is no need to repeatedly inoculate the work mixture;
rather after a single inoculation a system employing the present
denitration process can be run for extended periods without
reinoculation.
Broadly stated the present denitration process comprises
introducing aqueous tobacco extract along with necessary
additives into a vessel in which is a mixture containing suit-
able microorganisms while simultaneously withdrawing treated
extract from the vessel. By controlling the flow rate of
extract into and out of the system, the components within the
system and the conditions within the vessel, it is possible to
denitrate the extract without depletion of the microorganisms
and thus to effect treatment on a continuous basis.
The metabolic pathway employed in assimilatory
denitration can be represented as follows:
8 (H) + H NO3 ~ NH4 + OH + 2H2O
- Such assimilatory denitration thus involves the use of nitrate
as a nitrogen source to build up cell material.
In the practice of the present invention, micro-
,~
.
; - 8 -
~' _
.,. . . ~.
j~ 1:
3S7
I ¦' ganisms capable of assimilatory denitration must be employed.
various yeasts, particularly Candida yeasts, are capable of
nitrate assimilation. Among the Candida yeasts, the Candida
utills NCYC 707, 321 and 359 strains, the Candida utilis DSM
70167 strain, which is the same as the NCYC 359 strain, and
the Candida berthetii CBS 5452 strain have been found partic-
¦ ularly effective in the practice of the present invention.
Microorganisms, such as Enterobacter aerogenes, particularly
~ ~nterobacter aerogenes ATCC 13048, which is the same as the
O !i DSM 30053 strain, may also be employed in the practice of the
'I present invention.
j! These cultures are available at the culture banks
indicated by the abbreviations. The meaning of the abbreviations
l is as fol~ows:
S I NCYC Nation Collection of Yeast Cultures
¦ Brewing Industry Research Foundation
¦¦ CBS Central Bureau of Mold Cultures
, ATCC American Type Culture Collection
~ .
, DSM German Collection of Microorganisms
3 1, Tables I, II, III are descriptions of the cultures.
¦l The Candida yeasts and Enterobacter aerogenes ATCC
¦l13048 are characteri2ed in Tables I-III.
¦; ` Table I !;
¦ Çharacterization of Candida Yeasts
i ¦~Plasmodium or pseudoplasmodium-; motile cells-; ballistospores-;
¦ no~olar budding-; bipolar budding-; budding on stolons-;
triangular-shaped cells-; teniform* cells-; short lived celIs
with slow growth on malt agar and strong acetic acid production-;
~formation of true mycelium-; formation of pseudomycelia+; red
or.orange-colored cultures-.
'`'.' ' ' ,
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11 2~33S7
1 ¦ Table II
. ' - Characterization of Candida utilis
. ... I and Candida berthetii
I Fermentation Candida utilis Candida berthetii
S i' . . NCYC 707 CBS 5452
. i NCYC 359
. . ¦ CBS 321
, ~
Glucose + +
Galacto~e. , - -
Sucrose . +
I! Maltose
.. i. Cellobiose - -
.10 ! Trehalose
. Lactose
.il Melobiose
Raffinose +
.j' Melezitose* -
ii Inulin : - -
il ' '
. ¦ Assimilation. .
. ~ . 1, glucose+/+, galactose-/-; L~sorbose-/-; sucrose+/-; Maltose+/-:
.I cellobiose+/+; trehalose+*/-: Lactose-~-; melobiose-/-;
., 11, .
. ! raffinose+/-; melizitose+/-; inulin+/-; soluble starch-/-;
., . . D-xylose+*/-: L-arabinose-/-; D-arabinose-/-; D-ribose-/-;
: L-rhamnose-/-: ethanol+*/-: glycerlne+!+; erythrol-j-;
. tibitol-/-; galactitol-/-: D-mannitol+-*/-: D-glucitol-/-;
~ .
¦ ~-methyl-D-glucoside+*/-: salicin+/+: DL-lactate+/-:
. ~. ¦ succinate +*/+*; citrate+/+*; inosi~ol-/-.
... .¦ Assimilation of potassium.nitrate+/+; growth in vitamin
frqe medlum+*j+, growth promoting vitamins thiamine; NaCl-
tolerance ~ (W/V) 6-8/6-7: maximum growth temperature in C
. 11 39-43/40-41.
- good
,,~",, ,., I ~ '' .
. .j * = weak
.,~ . li~- = not present
30 11
, . . . ;
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- ! !
i Table III
Characterization of Enterobacter
aerogenes ATCC 13048
Cellfo~ short rodis; cilia peritrichous; motility+;
sporeformation-; pig~entation-; gram reaction-; aerobic~;
1¦ anaerobic~; catalase~-; oxidase-i nitrite formation from ~i
¦¦ nitrate+; indole-; methyl red-; Vosqes Proskauer+; citrate+;
H2S-; urease-; gelatin-; lysine decarboxylase+; argininede-
i hydrolase-; ornithinedecarboxylase+; phenylalaninedesaminase-;
¦ m~lonate~; gas from glucose~; lactose+; saccharose+; mannitol+;
dulcitol-; salicin+; adonitol+; inositol~; sorbitol+; arabinose+;
.,
! raff~nose+; rhamnose~.
¦ The process of the invention is practiced by provid-
¦ ing a work mixture comprising tobacco extract, additives, and
microorganisms at conditions suitable for assimilation of
j nitrogen-containing compounds, specifically, nitrites, nitrates,
and ammonium compounds. For purposes of the present invention,
references to nitrogen-containing compounds are to be under-
stood to mean nitrogen-containing compounds which are aerobic-
ally assimilated by microorganisms. Denitration in turn is to
be understood as referring to removal of such nitrogen-containing
- compounds.
; The work mixture comprises suitable microorganisms
.. .. . .
in tobacco ~xtraat under conditions which promote aerobic
~5 assimilation of nitrogen-containing compounds. Generally, to
:, ;
I start assimilation 30-100 g of starter culture mass of micro-
; organisms are inoculated per liter of tobacco extract under
.~, .. . .
conditions favorable to aerobic assimilation.
.. . . . . ................................................... .
For optimum results and to avoid lag phase, a starter
. .
~ 0 ' i~ cultuxe which is in expone~tial growth phase, and preferably
., ,j .
:: , . .. .
'~ i! .
'!
l,
- --1 1--
. . .
., ! '
2~3357
I , late exponential growth phase, and which has been pregrown on
, to~acco exera~ct is employed. Such a starter culture of Candida
' j yeast can be prepared for example by inoculating tobacco
,' extract with 2 loops of yeast, incubating the inoculated yeast
S for 9 hours, employing 10 ml of the resultant solution as an
inoculum for 200 ml of fresh extract substrate and thereafter
"incubating for 15 hours. Typically the amount of such a
'~ , ,' starter culture used to inoculate the work mixture is su~ficient
' , ' to produce a final concentration of culture in mixture of at
'- 10 , least 0.5-1%.
' ,Conditions which promote effective aerobic assim-
,~,ilation are maintained in the work mixture during the practice
!~ of the process. Generally suitable conditions for Candida
.. j, . ............... .. . .
; ',1 yeasts are an aeration rate of 0.8 to 2.5 liters air/liters
,,'~ ~ ' , wor~ mixture/minute, a pH value within the range of 3.5 to 7.2
.
, , ',1 and a temperature at a point between 25 iand 37C which is
'" ' ', favorable for a'large proportion of nitrate elimination relative
, , , li to c,arbon added plus adequate agitation.
,'' ,, ' !l ; .
, ' In the continuous operation of the process, a sterile
., ~ , .................. .
! additive mixture made up of tobacco extract and additives is
- , ' add;ed to the work mixture at a dilution rate which does not
,';~ , " , exceed the growth rate of the microorganisms employed. This
; ',; dilution rate is measured as liters of additive solution per
,i 11ters work mixture per hour. GenerallyO dilution rates of 0.1
" !i'to 0.35 l/l/hr are acceptable. Sterilization of the additive
:i, '' mixture can be accomplished by heating.
i;. . ,: .
' ;'''' ' !' The additive mixture may be added as a single solution
~ l containing'the tobacco extract and additives or the individual
,' ' ma~erials may be separately introduced into the work mixture.
,,l, Overall the total additi~ns to t;he work mixture comprise 0.1 to
, -12-
l :
~ ~ 2~ )7
1 l¦ 7.5 grams nitrate/liter of total additive mixture depending on
¦¦ the microorganism employed and 1.0 to 10 grams phosphate/liter
¦l o~ total additive mixture, as well as a carbon source at a
¦ concentration sufficient to provide at least 16.5 assimilative
S ~I carbon atoms per molecule of nitrate added.
¦l During the practice of the process, a portion of the
.1
, worX mixture is continuously removed at a rate such as to keep
the volume of the work mixture constant. The withdrawn work
Imlxture may be further treated to remo~e the biomass therefrom,
¦ that is, the microorganisms are removed, whereby denitrated
extract is obtained which is of substantially the same composi-
tion as the original extract except for the removal of th~i
nitrate.
I, .
!l It is preferable to allow the starter culture to
¦I reac~ the exponential growth phase in the work mixture prior to
¦ commencing continuous operation of the process. However, aside
j, .
;~ ! from the one-time starting phase, whose products can be dis-
~ carded, the process can be operated on a continuous basis with
! maintainance and regulation of conditions for the effect desired
with little or no supervision. In contrast, various treatment
'l phases are required in a noncontinuing, charged, so called
- batch process and if mistakes are made w1th a batch process,
new conditions for assimilation have to be ~chieved, which
!.' '1.l could take hours. Production by batch techniques is, thus,
! costlier and requires more personnel than with the process
de;scribed in the invention.
. . .
~he aqueous tobacco extract employed in the process
, of the invention may be obtained in a conventional manner.
; ! O~e method comprises contacting tobacco with water in a 1:10
ratio, oommonl~ at elevated temperature. ~he insoluble tobacco
, I
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, i -13-
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~ . 112~357
1 II residue is thereupon separated from the aque~s extract by
suitable solid/liquid separation techniques, such as
centrifugation, pressing or the like. The insoluble residue
~ may then be dried or subjected to reconstitution. If necessary,
S ll the concentration of nitrate is adjusted for addition to
. - ....... ,-
the work mixture by evaporation or dilution of the tobacco
¦I extract.
¦ In contrast to batch methods, wherein the phosphate
I present in stem pool extracts is ~ufficient, phosphate must be
1 added in the practice of the present process. Thus the additive
!I mixture must contain phosphates, as well as a carbon source, in
I! amounts sufficient for cell growth and total nitrate absorption.
Typically 1.0 to 10 grams of phosphate/liter of additi~e
m'ixture and enough carbon source to provide at least 16.5
i' assimilative carbon atoms/molecule of nitrate added are adequate
' ¦' for additive mixtures containing 3-7.5 grams nitrate. Preferably
! the concentrations of carbon source and phosphate are such that
l, they are consumed during assimilation of the nitrate and thus
I! do not reach the final denitrated extract. However, higher
~l concentration5 of these materials can be tolerated in the
:¦ practice of the present in~ention.
, 1 The carbon source may be any material that will
; ! provide the necessary carbons in an organic form usable by the
- ¦ microorganism to assimilate nitrates, nitrites and the like.
j Va*ious carbon sources have been found suitable in the practice
of the invention. ~or example! glucose, dextrose monohydrate
¦, and beet lasse-~ have proven satisfactory. The carbon may
¦I a~so be derived from the acid employed to adjust the pH of the
11 work mixture, for example, from lactic acid. With the Candida
11
yea9ts glucose, ~ucro5e~ malto8e, cellobio8e, ethanol, glycer~n
i
. `' . i` ' " .'
.~' .. . . . . ..
` ! : -
~ 14-
~ 2~3.~7
- 1 '
1 lor citrate are all suitable carbon sources. With Enterobacter
;¦¦aerogenes, lacto~e may additionally be employed.
j Generally, 16.5 assimilative carbons are required as
' l¦an absolute minimum for assimilation of a molecule of nitrate.
S ¦~On the other hand, the amount of carbon source is preferably
kept as close as possible to the threshold, since any excPss
will rema'in in the finaI denitrated extract. The threshold is
generally about 20~5 carbons/nitrate molecule. With good
¦aeration, a maximum of about 6.2 g~l NO3 can be assimilated
' 10 Iwith a 4% glucose solution. In general, with nitrate levels of
'¦ 3~7 5 g/l added to the work mixture, a concentration of 2.4- !
¦ 6% glucose is required when employing Candida yeasts.
"~ ' ' ' '¦ ' ; In the practice'of the invention, temperature affects
the amount of carbon required. At temperatures of about 28-
' 30C, minimal amounts, i.e., about 16.5 carbons, may be used.
~' Outside this'temperature range, the amount of carbon must be
' increased. Further temperature and ~rowth rate of the micro-
organisms are directly reIated. Thus higher temperatures favor
increased growth of microorganisms. However, increases in
~0 temperature also increase the fermentation rate, with resultant
' alcohol formation rather than growth. The rate of fermentation
may be checked by measuring ethanol formation during the process.
~ Temperature~ which minimize fermentation while maximizing
''' ' ¦ growth are thus preferred. In the case of Candida utilis, 30C
Z3 1 is the preferred temperature.
~ ' . . . . .
~ ' To regulate and maintain pH, acids and/or bases are
employed in the work mixture. Ortho-phosphoric acid and/or
potassium hydroxide-are preferred for this purpose. The agent
employ~ed to adjust pH may also be the phosphate or carbon sourcè,
for ~xample, phosphoric, lactic or citxic acid or mixtures thereo~.
.,` , . ..; , ,` " , "
. ." '',''
. I -15-
13.2~ S7
-J~ ~, where phosphoric aci,~ is employed to regulate pH, no
other addition of phosphate in the additive mixture is required.
Aeration of the work mixture is generally at a rate
which is sufficient to avoid fermentation while favoring
a5similation. Generally a rate of 0.8 liters air per liter
' work mixture per minute is the threshold aeration rate required
to avoid fermentation where maximum carbon levels are employed.
i,~verall, aeration rates of between 0.5 and 2.5 are suitable
in the prac'tice of the process, with rates of l.0 to 2.0 being
! p'articularly effective.
In order to overcome the effects of air injection,
''it may be necessary to employ a mechanical foam breaker or
antifoam agent. Paracum 05/12A and 24/sw have both been found
'satisfactory. A~dition of 225 ppm to the work mixture is
" adequate hut levels of 250 ppm are preferred to ensure trouble-
free operation.
' The precise conditions employed in the ~ractice of
.. ..
' the present invention will depend upon the precise organism
'employed. In general, when two of the three conaitions for
jaerobic assimilation are optimized, the third variable can be
' changed empirically. Further, it should be noted that since
the nitrate extracts being treated are solutions of naturally
occurring products whose components vary, optimum conditions
are not always the same, but will vary within the ranges
indi~ated. For example, in the case of Candida utilis NCYC
~70?, optimum conditions for the practice of the invention in
.
' ''the~treatment of some extracts are an aeration rate of 1.5
~l/l/min., a temperature of 30C and a pH of 5.5. Thus, very
good results are obtained with an aeration of 1.5 liters/liters
, /m,inute, a pH'value of 5.5 and a temperature between 26 and
. ' . ' ' , . . .
~ . ' , . ,
. .. .
,j , .
:. ~,i . . ' '
.
~ ~z93s7
i .
37C; with an aeration of 1.5 liters/liters/ minute, a temperature
,of 30C and a p~ between 3.9 and 5.5; or with a temperature o~
~30C, a pH~of 5.5 and aeration between 0.5-1.0 liters/litersf
iiminute.
; A denitration system emplo~ing the process of the
j invention is depicted in the flow diagram of FIGURE 1.~ The
;, .
..
~ reference numbers refer to the denitration stages as follows: .
J,
1. Tobacco supply.
2. Water tank
. 10 !l~ 3. Supply for additives
.~ il 4. Washer with partition
¦l'5- Mixer
.. 1' 6. Sterilization section
! 7. Sterilization section
. 8. Mixer
. ,. , ' .
~ . 9. Dosage pump
;~ i! 10. Work container (possibly fermentor)
'' 12. Pasteurization
! 13- Centrifuge
'. ' !' .
' 14. Trea~ment section
,. ~ . ; . . .
. ' 15. Device for readding of materials
17. pH regulator
.,, ~...... . .
18. Temperature regulator
; " 19. Aerator
! ' 20. Stirrer
. :1'. ' ' .
; . ll 21. Device for adding antifoaming agent
22. Section for reconstitution
23. Drier
The material to be treated, for example, tobacco
30 li stem~, i8 added ~rom tobacco supply 1 and mixed with water
. , ' ' .
,
.', . ' , .
-17-
. , '
357
!i 1 water tank 2 in washer 4. The soluble components are
Il separated from the insoluble tobacco residue. The insoluble
: ¦ residue.is passed to drier 23 or reconstitution stage 22. The
. ~ extracted soluble'components are conveyed to 6 where sterilization
.by heating' and thereafter cooling takes place. Specifically,
; ¦ the treatment in sterilization sections 6 and 7 may cons~st of
. . ¦ preheating to 100C, sterilization of 110C for over 40 minutes
. . I and cooling to 30C.
: The necessary additives, mostly phosphate and glucose,
. -t~avel from supply 3 with water to mixer 5 and as solution to
section 7, where they are sterilized by heating and thereafter
.~r . co:olea. The solutions from the two treatment sections 6 and 7
.are.mixed in mixer 8 and are by way of dosage pump 9 transferred
'into work vessel io. To start', fermentor 10 may con~ain a
. !
woxk mixture, comprising the product .solution with the necessary
. I additives and an inoculum of the desixed microorganism, from
t which all~the nitrates, nitrites and ammonium compounds will
ge~exally be eliminated after about 8-20.hours, whereupon the
' ¦l continuing process can be started b~ the dosage pump 9 at the '
l 1, rate desired for dilution ana regulated in such a way as to
..... .
ke~p the volume-of the work mixture in fermentor 10 constant.
. ; 'I According to the~products and microorganisms used, the working ',
il conditions are re~ulated in such a wa~ as to totally eliminate
!~- . .
. nitrates, nitrites and ammonium compounds contained in the
; . . ;j . . ,
S '; pxoduct.solution and to completely use all additives from
. ; ~. mixer 5 during this assimilation The treated woxk mixture in
'.i- fermentor 10 is removed. The biomass is removed from the
treated work mixture in centrifuge 13 and may be saved for
further usage. If necessary~ the treated ~ork mixture may be
. pa~teurizec~ in ~e~tio'n 1~ as shown in FIGURE 1 or l?asteurization '.
,
.. ',' ' ' , .
' :, ;
--18--
i! , .
5"7
the liquid portion resulting from biomass removal in section
13 may be effected. The remaining treated liquia is conveyed
to treatment section 14 as final solution for concentration,
!l as by evaporation. This final solution, containing most of
ji the components of the product solution, except the nitrates,
'I nitrites and ammonium compounds may now be used in any way.
! ;The solution may for example, be sprayed onto the dried or
reconstituted tobacco residue with de~ice 15 for readding
¦ materials. The reconstituted product resembles tobacco sheets.
1~ In a preferred mode the present invention comprises
i! extracting tobacco with water employing a l0:l water to tobacco
~i1 ratio at 90C for-60 minutes. The extract thus formed is
¦~ separatea from the insoluble to~acco residue. If necessary,
ll the nitrate concentration in the extract is adjusted to the
i desired level by conventional means such as dilution or evapora-
tion. The extract at a dilution of 3 to 7.5 g NO3/liters, pxefer
ably 4.5-5.5 g/l and most pre~erably 5 g/l, is thereupon combined
with sufficient K2HPO4 to give a phosphate concentration of
l l-l.5, preferably l.25, and glucose is added to a concentration
~ of 4%, along with 250 ppm antifoam, such as Paracum 24/sw.
li The pH is adjusted to 5.5 employing KOH. The mixture may then
~, be sterilized at 110C for forty minutes. Alternatively, the
, extxact and additives may be separatel~ steri1ized prior to
j, mi~ins.
il, The sterilized extract solution containing the additives
'~ ;s thereupon introduced into a fermentation vessel containing a
work mixture at a rate of 0.18-0.22 l/l/hr, preferably 0.2
l/1/hr. The work mixture contains a suitable microor~anism and
~,~ i5 pre~erably a starter culture of Candida utilis NCYC 707
yeast in exponential, most pref~rably lat~ ex~onential, c3rowtn
. 1,~ .' , . I
.. ,. . , :
jl !
;. ,1 . 19
i,, . ' ' .
'' ~!! - .
¦IE ~ which has been built up as above described. The pH of the
work mixture is maintained at about 5.5 + 0.3 preferably by addi-
tion of a mixture of 9 parts lactic acid to 1 part o-phosphoric
¦acid and/or KOH. The temperature of the mixture is maintained
~at 30 t 3C. The vessel containing the work mixture is aerated
,at a rate of 1.4-1.6 and preferably 1.5 l/l/min. and the ~ixture
!~is a~itated In smaller vessels it may be desirable to shut
,~r ~,off the air for one minute every two hours, whereby the pressure
l~is xeduced and the condenser on the outgoing air is purged.
- : ., ,
This can be accomplished by means of an electromagnetic valve
¦lcoupled with a time on the incoming air. Such purging avoids
': ' '~1 .
jlwetting of the sterile filter. Such purging is generally
unnecessary when working in larger fermentors, as for example,
~,when a 500 1 working volume is employed in a 750 1 fermentor.
! Simultaneously wi~h and at a rate equal to the intro-
jlduction of the sterilized solution, a portion o~ the work mixture,
- .'i.e., treated extract is withdrawn from the fermentation vessel
'50 that the volume of work mixture remains constant. The
treated extract is thereupon pasteurized, separated from the
~0 ~ iomass and concentrated. The $hus ~enitrated t concentrated
extract may then be applied to the dried and/or reconstituted
,, . . . , .~ i
eins~luble tobacco residue. Employing the above procedure, the
process of the invention has been practiced continuously fox ~ive .
weeks With production of 2400 liters denitrated extract per day
. . . jl , . . .
: ~,which is equal to one-fifth of the ~olume of the fermentor employed~
jpei hour, i.e. r 100 liters fermented denltrated extract per hour~ !
~ here Enterobacter aerogenes ATCC 13048, or other
acterium, is employed the conditions of the work mixture are
,adjusted to a pH of 5.5-8.0, preferably 7~0, and a temperature
IOf ~0-40~Ct prefera~ly 3?~C, and the process is operated at
. ' ' ~, . . . .
', ' '
;' ~20-
. '' , ' ' ' , ' ' .
~ , ,_ . .. _.. , . - - . . .
~12~3~C~7
. aeration rate o 1.0-3, preferably 2 l/l/min., a dilution
rate of 0.1-0.25, pre~erably 0.2 l/l/hr., with the addition
mixture containing 0.1-7.5 g nitrate/l, preferably 5 g/l.
¦ T~e invention is preferably used in treatment of
tobacco extracts, bu~ is not limited to that usage. Elimin-
!l ation of nitrate~, nitrites and ammonium compounds from foods
and other consumer items may also be desirable. Where these
! materials are in liquid form, they may be used as the nitrate
I solution for treatment in the practice of the invention.
~ Otherwise an aqueous solution ~an be obtained by washing,
' which solution, following denitration, may be recomb'ined with
' the insoluble fraction of the material to form the final
denitrated product.
In the case of tobacco, the work conditions can be
il gauged by the nitrate concentration of the product solution.
~ To determine workLng condition's for foods and other consumer
¦ll items, thè'concentration of the sum of all compounds to be
¦ eliminated, i.e~., nitrates and nitrites and ammonium compounds
' should be considered. This total concentration of these
' materials in the overall additive mix'ure should be between 3
, and 7.5 g/liter. The remaining parameters may be the same as '
jj in trea~ment of tobacco. Thus,' although the invention has
¦ been described'in terms of its application to tobacca, it
i may--apart from the limitations described be~ore--ju9t as well
l be applied in the treatment of foods ad other consumer goods. I
The following examples are illustrative of the invention.
.
, Examples 1-10
; Tobacco stems were'extracted with water and the
resultant extracts were treated with Candida utilis NCYC 707
! according to the process'of the inven~ion using the conditions
'~
' ' 1 . . .
!i ,
-21- I
. . , i
.' ' .
r~ L12~ 7
~ ecified in Table I~. The results are set forth in Table IV.
Il `u" indicates an amount, which is not detectable using normal
Il analysis conditions; it is smaller than 10 ppm in the case of
li carbon and phosphate and is less than 1 ppm for nitrates, . I
nltrites and ammonium compounds.
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I Example 11
¦ Seven tobacco extracts were prepared by separately
j washing tobacco stems and by-products with water at a 1:10
tobacco to water ratio'and combining a stem extract with a
S j;by-product extract. With appropriate installations, it would
be possible to efect the extraction, as well as the denitration,
¦'-on a continuous basis. The nitrate levels of each tobacco
: ~ extract are set forth in Table V. To each tobacco extrac~ were
¦'added glucose, KHzPO4 and Paracum ~4/sw antifoam as indicatea
¦ in Table V. The ph was ad~usted to 5.5 with KOH. The extract
' ' ¦ and adaitives were sterilized at 110C for 40 min.
_ _ ,
' The work mixture comprised Candida Utilis 707, lactic
acid, KOH and Paracum-24/sw and had a pH of-5.5. The 14 1 fer-
; mentor, which was employed, was equipped with an electromagnetic
vaive on the incoming air coupled with a timer. ~rhe incoming
air wa's shut off for 1 min. every two hours to thus pur~e the
' conden~ser on the outgoing air.
. ' About 1700 1 of extract were denitrated on a continuous
¦ basis except for the first two weekends where the system was
¦ cooled down with agitation reduced to 300 rpm and air flow
¦ reduced to 30%. After the first week there was no super~ision
" ¦ over the wee~ends or durin~ the nights. Except for a problem
' " ¦ with the weight contxol'system, which resulted in the fermentor~
;¦ being empty one morning, the operation ran smoothly.
During denitration 200 ml of lactic acia were consu~ed
I per 3 kg of extract. This is probably explained by the fact
'' ' l that the acid was being used by the microorganisms as a carbon
~ source~
¦l The biomass was removed frcm the denitrated extract
¦ ~y centrifu~ation. The resulting 1~7~ g of extract containing
. . .
." ' ' .1
!i
i! - -24-
.. l'
.
"
lZ~3
.
I!
1 1l 3% tobacco solubles was thereupon concentrated to give an
,. . . .
¦i average concentration o~ 39.06 tobacco soluhles and reappliea
,1 . .
,¦ to ~ried tobacco stems.
¦ The average values of the composition of several
! sampleswere measured after extraction, after steriliza~ion of
; ¦ the combined extract and additives and following concentration
of the denitrated extract. These values are set forth in
I
I Table VI.
¦ . , !
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129357
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9357
~ '.' Table VI
, .
. ExtractConcentrated
¦' . and Denitrated
- Extract Additives Extract
,I PO4+ g/l 0.4 2.7 24
S ,. SO4 g/l 1.32 21 ~,~ 23
. Il K~ g/1 6.8 10.75 70
- il Ca2~ mg/l 68g 174 100i~
ll Mg2+ . mg/l 269 230 2900
. I .
. . , e~hanol mg/l 2.8 2.8 5.4
I methanol mgjl 13~5 20 ~8
Il acetone mg/l 2 . 6.4
.~ . ijl acetoine mg/l 1~3
,¦ total C /oo 14.4 28.1 140
'¦ NO~ -g/l 2.30 2.44 0.4
NO3-N g/l 0.52 . 0.55 0.0g
.I RS . g/l 4.24 41.8 5.7
'l ~ " ' . . ,
NH3-N g/l 0.23 0.24 0
I TA g/l . 0.52 0.52 6~2
.- .i . . .
. Example 12 .
:20 I A continuous one week pilot plant trial was carried
.. . , .
: ; ~ out in a 750 1 fermentor ~working volume 500 1) with tobacco
stem extrac~. Operating conditions ara given in Table VII.
" The stems were continuously washed in a screw
extractor at a stem to water ratio of 1:10. The extraction
2$ !~ .
was carried out at 90C and the tobacco extract (out of the
; extxactor) to water (into the extractor) ratio was 0.74. The
!, tobacco extract was then sterilized by pumping it through 3
' i heat exchangers: the first to pre-heat it to 110~C, the second
., ; . .
.; ~. to hold it at that temperature for 40 minutes and the third
, 30 il
. 1 to cool it down to room temperature~ Analytical values are
' ' ~' ' - ' , .
! .
Ij .
'. .. .
;! . .
... . .. . . . .
,~ l~Z9357
~ . .. .
given in Table VIII.
A dextrose solution was prepared batchwise, but then
continuously pumped through a second line of 3 heat exchangers
i! for sterilization using the above conditions. The two flows,
S l¦i.e., sugar solution and tobacco extract, were then r~gulated
¦¦to the desired sugar concentration in the tobacco extract and
,~jthen pumped into the fermentor? Nitrate and sugar values are
jlgiven in Table VIII.
¦ Before the start o~ continuous operation, the
10 ; fermentor was filled with 480 kg of tobacco extract, 20.2 kg
of dextrose,''2.4 kg of KH2P~4 and 120 ml of an antifoaming
,j .
¦agent, and then sterilized at 120C for 40 minutes.- After
¦the fermentor haa been cooled down, it was inoculated with
13 1 of a starter cultur'e of Candida Utilis 707 grown in tobacco
~extract. After 12 hours there was no more sugar or nitrate
¦in the batch and the yeasts were in the exponential phase.
¦¦At this-point continuous operation was started. The operating
¦ conditions are given in Table VII. The pH regulation was
'" ' ''I don~ with phosphoric acid at 25%. The fermentor was equipped
'20 ; ¦ with a mechanical foam separator, a turbine aeration/agitation
. system, and a weight control system.
'~ ' The continuous stream of fermented extract leaving
~ ¦ the fermentor was centrifuged to remove the biomass and then
."~.' ,'. I .
'' '! pasteurized before being concentrated.
' ' All these operations except for preparation of the
sugax solution were carried out continuously.
: " , . . ,~ .
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