Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
S P E C I F I C A T I O N
The present invention relates to a process for the
production of fats and oils, and particularly fats and oils
rich in triglycerides, from microbial sources.
It is well known that fats and oils can be
produced by cultivating an oil-synthesizing microorganism,
including algae, bacteria, ~olds and yeast. Such micro-
o~ganisms synthesize oils and fats in the ordinary course of
their cellular metabolism. Extensive research has been
carried out in an effort to identify microorganisms, media
and conditions which would permit economically practical oil
production.
One field of production of fats and oils by
ferméntation which has received particular attention is the
field of producing cacao butter substitutes. Cacao butter
is a naturally-occurring substance which contains large
quantities of 1,3-disaturated-2-unsaturated triglycerides.
These triglycerides include l-stearoyl-2-oleoyl-3-palmitoyl
triglycerides and 1,3-dipalmitoyl-2-oleoyl triglycerides.
A process for producing triglycerides rich in the foregoing
compounds is described in U. S. Patent No. 4,032,405,
granted on June 28, 1977.
As described in this patent, a cacao-butter
substitute is produced by cultivating a ~icroorganism from
the genus Endomyces, Rhodotorula, Lipomyces or Rhodospordium
under aerobic conditions, followed by collecting the cells
and isolating the fats and oils rich in 1~3-disaturated-2
~nsaturated triglycerides from the cells. The medium
461~
-- 2 --
employed in the fermentation process of the foregoing patent
generally includes a source of assimilable nitrogen, and a
carbon source preferably in the form of an aldose or a di- or
polysaccharide. The resulting cells are collected and from
them is isolated a mixture of the fats and oils which are
rich in l,3-disaturated-2-unsaturated triglycerides.
Improvements in the process as described in the
foregoing patent are described in our Canadian patent no.
1,139,692 issued January 18, 1983. As described in that
patent, it has been found that the yield of the fats and oils
can be increased and the distribution of the particular fats
and oils can be controlled when the fermentation medium
includes a carbon nutrient source in the form of one or more
fatty acids containing between 10 and 20 carbon atoms. For
example, it has been found that the ratio of saturated to
unsaturated acid groups of glyceryl oils may be controlled by
employing in the fermentation medium the very acids which
form the fatty acid portion of cacao butter, namely palmitic,
oleic and stearic acids.
While the process described in the foregoing patent
represents a distinct improvement in both the yield and acid
distributions in fat and oil fermentation processes, there is
nevertheless room for further improvement. It is thus
desirable to investigate the factors affecting the production
of triglyceride oils produced but also the control of the
distribution of the acid component of the triglycerides
themselves.
It is accordingly an object of this invention to .......
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provide a process for the production of fats and oils by
fermen1:ation in which the yields of such fats and oils are
dramat:Lcally increased.
It is a more specific object of the present invention to
provide a process for the production of fats and oils, and
particularly fats and oils rich in triglycerides from microbial
sources, wherein the yield of the desirable saturated fats and
oils is increased with shortened reaction time while the yield
of the less desirable unsaturated fats and oils is decreased.
The concepts of the present invention reside in a process
for the production of fats and oils, and particularly fats and
oils which are rich in triglycerides, wherein yeast cells which
are capable of synthesizing the fats and oils are cultivated in a
medium containing an homogenized emulsion of at least one fatty
acid containing 10 to 20 carbon atoms and containing an
emulsifier having an HLB above 15. It has been found that the
use of an homogenized emulsion of the fatty acid component of the
fermentation medium increases the availability of the fatty acids
during their assimilation by the yeast cells to thereby further
increase the yields of the fats and oils produced while control-
ling the acid distribution of the acid components forming the
triglycerides.
The process of the present invention is particularly well
suited for use in the production of fats and oils of the type
which are predominant in cacao butter. It has been found, in
accordance with one embodiment, that the production of such
oils can be significantly increased ....
1~7~6~.~J
where the fermentation medium is formulated to include an
emulsion of palmitic, oleic and stearic acids.
In accordance with one embodiment of the invention, it
has been found that the use of a desaturase enzyme inhibitor
promotes a higher ratio of stearic to oleic acid radicals
present in the resulting triglyceride oils. Without
limiting the invention as to theory, it is believed that the
desaturase enzyme inhibitor serves to minimize the effect of
intracellular desaturase which in turn prevents desaturation
of the stearic acid. Thus, the use of the desaturase enzyme
inhibitor results in increased stearic acid levels found in
the resulting triglycerides.
While the present invention will be described hereinafter
with reference to the production of fats and oils of the type
which are predominant in cacao butter, that is triglycerides
containing 1,3-distearoyl-2-oleoyl triglycerides, l-stearoyl-
2-oleoyl-3-palmitoyl trigl~cerides and 1,3-dipalmitoyl-2-
oleoyl triglycerides, it will be understood by those skilled
in the art that the concepts of the present invention may
likewise be used in the production of other fats and oils by
fermentation.
The microorganisms useful in the practice of this
invention may be characterized as oil synthesizing yeasts;
such yeasts are well known and available to the art. For
example, a number of them are described in U.S. Patent
No. 4,032,405. Particularly preferred for use in the practice....
~'7'~L6~9
of this invention are species from the genus Rhodosporidium,
Lipomyces, Candida, Endomyces, Saccharomyces, Rhodotorula,
Trichosporon or Torulopsis
Such oil-synthesizing yeasts are well known and
can be isolated by conventional techniques from native
sources such as leaves, vegetable stems and the like. It
is generally more convenient, however, to obtain such yeasts
from various culture storage deposits including, for example
the American Type Culture Collection. For economic reasons,
it is generally preferred to employ an oil-synthesizing
yeast which has a tendency to synthesize snd store large
amounts of oils. Yeasts having the ability to accumulate
20% oil, and preferably at least 30% oil, on a standard
culture medium (such as glucose, ammonium salts and
minerals) are generally preferred.
The growth and/or fermentation medium providing
nutrients for the cultivation of the particular yeast
species to employ depends somewhat on the partic~lar yeast
selected for use in the process of this i~vention. In
general, such media are dilute aqueous basic solutions
containing carbon and nitrogen nutrient sources, generally
in amounts less than 6% by ~7eight based on the weight of the
medium. Preferred media are generally adjusted or buffered
so that the p~l ranges bet~een about 4.0 and 9.0, and
preferably 5 to 8.5, as is conventional for optimum yeast
cultivation.
As the nitrogen nutrient source, use c2n be made
'~
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~ 1 7~6~
o~ any of a variety of conventional nitro~en-containing
compounds frequently used as nutrients for microbial growth.
Preferred nitrogen compounds include asparagine, glutamine,
peptones and the like. In addition, other nitrogen-
containing compounds such as a~monium salts and urea may
likewise be used.
In general, the nitrogen nutrient source serves to
promote growth of the yeast, while the carbon nutrient
referred to above serves to promote fat accumulation in the
yeast cells. Thus, high nitrogen-to-carbon ratios are
useful in pro~oting growth of the cells while high carbon-
to-nitrogen ratios maximize fat accumulation.
One nitrogen-containing nutrient which is
particularly well suited for use in the practice of this
invention is cornsteep, the aqueous liquor formed in the
conventional corn-wet-milling process in which dry corn is
soaked in warm dilute sulfuric acid. Corn~teep is composed
of about 25% by weight of crude protein (8Z nitrogen by
weight) as well as small amounts of ash, sugars and other
beneficial culture constituents. While cornsteep can be
used alone as an inexpensive but yet com.plete nitrogen
nutrient source, it can be formulated with other
conventional nitrogen nutrient sources well known to those
skilled in the art.
The medium should also include any one or more of
the known essential metabolic mineral salts, including the
salts of potassium, sodium, calcium, magnesiu~, iron or the
(
~ 7~6~
like. In addition, secondary nutrients such as vitamins and
amino acids are like~ise desirable, particularly where the
cultivation period for the yeast is extensive.
The fer~entation medium employed in the practice
of this invention also contains, in accordance with an
important concept of the invention, a carbon source. As has
been described briefly above, the fermentation medium should
contain a predominant a~ount of one or more fatty acids
containing between 10 and 20 carbon atoms. Because it is
believed, again without limiting the invention as to theory,
that the yeast cells utilize the fatty acids in their
metabolism, it is preferred that the fatty acid content of
the fermentation medium constitute at least 10%, and
preferably 40~ or higher, of the total car~on source. In
that way, the fatty acids, to the extent they serve to
modify the metabolism of the yeast cells to produce
triglyceride oils having a particular fatty acid content,
are not masked by the presence of other carbon nutrient
sources in the fermentation medium.
The fatty acid or acids e~ployed as the carbon
source in the practice of this invention may be obtained
from any of a variety of known sources. For example,
palmitic acid (C16:0), stearic acid (C18:0) or oleic
acid (C18:1) can be obtained commercially, either in the
form of the free acid or salts such as the sodium salt.
These more com~on fatty acids can be employed alone or in
r mixture with others. Polyunsaturated fatty acids, such as
linoleic acid (C18:2), linolenic acid (Clg:3), and other
1~1 7'.-~6~
fatty acids containing 16 to 20 carbon atoms may also be
obtained in pure form but are more readily available in the
less expensive form of commercial mixtures, such as soap
stock.
The composition of the fatty acid employed is
important to the extent that each fatty acid causes a unique
type of shift in the oil-synthesizing metabolism of a given
yeast species. When use is made of a mixture of fatty
acids, their combined effect is an interaction to result in
the metabolic mixtures of triglycerides containing the
various fatty acids present in the fermentation medium.
However, accurate prediction of the precise yieid
in oil composition to be obtained from any particular fatty
acid carbon source is largely empirically ~ased.
Conventional analytical procedures permit the determination
of the yield in composition of oils produced from any
particular carbon sources, and hence routine experimentation
permits the ready identification of fatt~ acid carbon
sources suitable for the production of any particular oil.
Some generalizations in the form of general rules
have been determined, however. For example, the presence of
a fatty acid of any given carbon length ~n the carbon source
ordinarily results in the increase in the proportion of tri-
glyceride esters containing that fatty acid as a component
of the triglyceride. Similarly, the degree of saturation
and/or unsaturation (and particularly polyunsaturation) in
the oil produced is directly related to ~he corresponding
~ 7~
saturation level of the fatty acid composition employed 8S
the carbon source. Thus, the use o palmitic, oleic and
stearic acLds as the carbon source promote the formation o
oils which closely approximate those existing in cacao butter.
The conditions under whlch the yeast is cultivated
to produce fats and oils in accordance with the process of
this invention are not different from those generally
employed in prior art fermentation systems. In general, the
yeast employed in the practice of this invention to produce
such fats and oils are generally the same as prior art
processes employing the same type yeast species.
The fermentation medium has nitrogen-containing
nutrient such that the amount of nitrogen present in the
medium ranges from 0.005 to 1% nitrogen by weight, while the
carbon nutrient present in the fermentation medium generally
ranges from 0.1 to 5% carbon by weight. The temperature at
which the fermentation is carried out is generally within
the range of about 20 to 40 C, with higher temperatures
within that range favoring the production of saturated oils
while lower temperatures within the range ~avoring the
production of unsaturated oils.
Similarly, oxygen may have some effect on the
growth o the yeast cells. In general, it has been found
that aerobic cultivation of the yeast cells increases the
final yield of the oil produced by the microorganisms.
!
Once the fermentation has been allo~ed to carry
~7~
-- 10 --
out for the desired period of time, generally for one to seven
days and preferably two to five days, the yeast cells are
separated from the fermentation media by conventional means and
their oil content removed. For example, the cells can first be
subjected to rupture by, for example, freezing or hydrolysis,
and then the oil extracted from the debris with a suitable
solvent, preferably a volatile solvent to facilitate subsequent
removal of the solvent from the oil.
As noted above, it is an important concept of the invention
that the fatty acids present in the fermentation medium be in
homogenized emulsion form. That is preferably accomplished by
addition to the fermentation medium of an emulsifier which is
compatible with the fatty acids employed and which does not
adversely affect the metabolism of the yeast cells. In general,
emulsifiers employed in the practice of this invention are ionic
and non-ionic emulsifiers having an HLB above 15.
Preferred for this purpose are emulsifiers in the form of
fatty acid derivatives of sorbitol and sorbitol anhydrides.
Particularly preferred are non-ionic emulsifiers such as those
marketed by Atlas Chemical Industries Inc. under the trademark
"Tween", which are polyoxyethylene derivatives of fatty acid
partial esters of sorbitol anhydrides, and those marketed under
the trademark "Span", which are fatty acid partial esters of
sorbitol anhydrides. Both types of emulsifiers are approved by
the FDA for food use; it has surprisingly been found that they
do not adversely affect the metabolism of the yeast cells in
the formation of fats and oils.
!
, 11'7'~6~
ln general, only enough of the emulsifier as is
sufficient to emulsify the fatty acids present in the
fermentation medium need be used. In general, that amount
ranges from 0.~ to lZ based on the weight o~ the
~ermentation medium. The emulsion is preferably produced by
adding the emulsifier to the fatty acid or fatty acids and
then providing suficient agitation to produce a
substantially homogeneous ~ermentation medium, either with
or without the other components of the fermentation medium
having been added at the time of the agitation.
' In the preferred practice of the invention, the
emulsion is formed by heating the ~atty acid with a buffer
to a pH ranging ~'rom 7 to 9, followed by autoclaving the
fatty acid to sterilize it if necessary. Then the,emulsi,fier
is added and the resulting mixture homogenized. l'he
emulsion ls next subjected to rapid cooling at a rate
sufficient to crystalize stearic acid particles of very
small sizes. It has been found in accordance with the
practice of the invention that particles sizes less than 10
microns are particularly suitable to insure- that the fatty
acid or acids are utilized effectively in the fermentation
process.
As the carbohydrate, use is preferably made of a
car~ohydrate selected ~rom the group consisting of aldoses
(e.g., glucose, hexose, pentose, etc.), disaccharides such
as maltose, sucrose, etc. and oligosaccharides, and
preferably oligosaccharides derived ~rom the hydrolysis of
starch. Glycerol may likewise be advantageously used.
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~ 61~
In accordance with another embodiment of this
invention, it has been found that it is fre~uently desirable
to include in the ~ermentation med1um a desaturase enzyme
inhibitor. As is described above, and without limiting the
present invention as to theory, it is believed that the
desaturase enzyme inhibitor serves to minimize the e~ect o~
desaturase enzyme during the fermentation, and thus tends to
increase the ratio of saturated oils to unsaturated oils.
One such desaturase enzyme inhibitor which has
been employed is sterculic acid, the cyclopropanoic
derivative of stearic acid found in cotton seed oil. It
will be understood by those skilled in the art that other
inhibitors may likewise be used. Generally, the amount o~
such an inhibitor is an amount su~icient to inhibit the
desaturase enzyme, and is normally within the range of 0.001
to 0.5% by weight, and preferably O.Ol to 0.2% by weight.
Having described the basic concepts o~ the present
lnvention, reference is now made to the following examples,
which are provided by way of illustration and not by way of
limitation, of the practice of the present invention. In
those examples, all o~ the percentages are percentages by
weight unless otherwise indicated.
EX~;PL~ 1
This example illustrates the practice o the
present invention in utilizing stearic acid.
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.
~7~6~
An emulsion was prepared by $irst heating one gram
of stearic acid to about ~0 C and then mixing with it a
solution of potassium phosphate having a pH of 6 to 7. The
resulting mixture is then heated to 120 C for 15 minutes to
sterilize the fatty acid. l~ereafter, .01% of a gram of the
emulsifier Tween 20 was added, and the resulting ~ixture was
-subjected to a quic~ cooling to precipitate stearic acid
crystals having sizes ranging from 1 to 10 microns. The
resulting milky suspension had a pH of 6 to 7, was stable
and exhibited no coalescense. That emulsion was then
blended with a fermentation medium so that the resulting
fermentation medium had the following overall composition:
Peptone 0.5%
Yeast extract O.lÆ
Glucose 2.0%
K2HP04 0 . l'Z
Antibiotic 10 g/ml
Emulsifier (Tween 20) 0.01%
Stearic acid 1.~
H20 100 ml
The composition had a pH of 5.5 to 6Ø
The fermentation medium (Sample ~) was then..
inoculated at 28 C ~7ith yeast cells of R. toruloides grown
on a nutrient medium containing 5% ~lucose, 5% peptone and
1% yeast extract.
At the same time, a second medium (SPmple II) was
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1~';"~61~
was formulated in the same manner, except that the amount of
gLucose was increased to 4%. Another medium, Sample III,
WBS prepared in the same manner, except that it contained no
~lucose and the stearic acid level was 4% by weight.
Samples II and III were inoculated with the same
inoculum at 28 C. The fermentation of Samples I, II and III
was allowed to continue in a shake flask at 200 rpm for 6
days at 28 C.
. Another sample (Sample IV) was formulated,
inoculated and fermented in the same way as Sample I, except
that the pH was adjusted to 7.5 after 2-1/2 days. The cells
from each were then harvested, and the oil recovered and
analyzed.
The following results were obtained.
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ls 1:17~ 9
RESULTS - EX~MPLE 1
Sample I II III IV
x x x x
Mg. of Neutral200 225217 388 407 350 469 542 %
Oil
~ Conversion
based on lipid20% 23%22% 39% 21% 17% 47 54.28%
% Conversion
based on lipid
CH2O 7% 8% 8% 13% - - 16~ 18
C:12 .2 .3 .3 .1 .1 .1 .1
C:14 .71.0 1.2 .4 .3 .6 .5
C:16:0 17.4~2.8 20.6 13.2 1015.4 13
C:16:1 4.21.0 4.4 .5 .3 .5 2.1
C:18:0 3229.8 24.3 49.2 4839.6 35.2
C:18:1 31.332.2 32.0 25.7 2531.4 35.2
C:18:2 5.71.3 6.3 4.0 9.9 4.7 7.4
C:20 .6 .6 .7 .8 .7 .7 .6
C:18:3 .9 .3 .7 .3 1.9 .6 1.4
C:22 .91.0 1.4 .6 .6 .8 .7
Unknowns 4.07.1 4.6 4.9 3.6 4.5 3.0
Total saturates54 58 52 65 59.758.3 51
Total 35.533.2 36.4 26 24.831.9 37.8
monounsaturates
Total
polyunsaturates6.6 1.6 7.0 4.311.8 5.4 8.8
Theoretical
Iodine 42.332 44 30 43 3748.6
. ~¢'`
619
The foregoing results show that the presence of
carbohydrates in the fermentation medium serves to increase
the levels of palmitic and oleic acid levels st the expense
of stearic acid levels. In addition, the adjustment of the
pH of the fermentation medium to a pH above 7 results in
increased conversion.
EXAMPLE 2
-
This example illustrates the importance of forming
the emulsifier in the fermentation medium used ln accordance
with the practice of the present invention.
In this example, several fermentation media were
prepared, except that the procedure described in Example 1
was varied except as follows: the fermentation medium
employed in this example consisted of 0.02% potassium
phosphate, 0.02% Tween 20 or Tween 80 which was employed
with stearic acid in accordance with the following:
Sample
A Stearic acid (no homogenation, no quick
- cool, no emulsifier)
B 0.02% Tween 20 ~no homogenation or quick
cool)
C Stearic acid homogenized (no homogenation
or quick cool)
D Stearic acid homogenized with Tween 80 (no
quick cool)
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~q7~619
E Stearic acid homogenized with Tween 20 ~no
quick cool)
F ~tearic acid homogenized with Tween 20 and
quick cooling
G Stearic acid homogenized at a pH of 4.5 but
with no quick cooling.
All of the foregoing samples were inoculated with
R. toruloides grown in a 5-liter fermentator at 28 C for 48
hours. The fattening phase was carried out on a shake
incubator at 32 C and 260 rpm. One gram of R toruloides
was added to 100 ml of lipid media. After emulsions were
prepared, visual observations were noted and reported as
follows: ,
Sample Observation
A Large chunks of stearic
B Same as A
C Small particles of stearic
D Same as C
E ~ame as C
~` - No particles observed
G Same as C
Just prior to harvesting from the lipid media,
microscopic observations were made with the following
results:
-17-
`~ ~,7461~
Sample Observation
A&B Little or no lipid accumulation
C-E 10-20% lipid accumulation
F 20-40% lipid accu~ulation
G 10-20% lipid accumulation
The visual observations to the foregoing samples
prior to inoculation show that samples A-E and G contain
vi~ual observation of particles, thus demonstrating that
they were greater in size than 1 to 2 microns. The micro-
scopic examination just pr~or to harvesting revealed that
thë greater the particle size, the less ~as the lipid
accumulation. Samples A and B were not homogenized, and
thus had the largest particles. As the particle size
decreased (i.e., Samples C through E, inc~usive), larger
cellular lipid globules were observed.
The analytical results are shown in the following
table:
Sample Neutral Oil (mg.)
A - (No homogenation) 50.2 mg
- (No homogenation - .02% Tween 20) 54.7
C - (Homogenation-no emulsifier) 103.0
D - (Homogenation - Tween 80) 100.1
~ - (Homogenation-Tween 20-no quick cool) 106.9
F - (Homogenation-Tween 20-quick cool? 139.7
G - (Homogenation-Tween 20-pH 4.5) 93.7
The results shown in the followlng example
-18-
~461g
illustrate that a good emulsion is essential to effective
utilization of stearic acid. The factors contributing to
the for~ation of a good emulsion are proper homogenization,
a proper level of emulsifying agent, a neutral pH and a
quick cooling in an ice pack. The use of an emulsifying
agent without heating or melting of the fatty acid with
homogenization and auick cooling is not particularly
effective, the data show, with fatty acid carbon sources
having high melting points. This is why the emulsion process
of the present invention was developed.
Of importance to the structure and meltin~
properties of cacao butter is the degree of unsaturation
in the B-position. Listed in Table ~ is the B positional ~6
data for a given sample. This is the sterospecific method
using pancreatic lipase. Luddy F. E. et al. JAOCS, Vol.
41, p. 693 1964.
-- 19 --
61g
TABLE ~æ D~
F~.C of triglyceride B-position
C:14 0.4 33
C:16 26.1 4.7
C:16:12.2 1.2
C:17 1.4 ---
C: unk0.4 .73
C:18 28.7 2.9
C:~8:131.5 72
C:18:27.2 16
C:18:31.0 1.5
C:22 - 3
The information demonstrates that the fatty
acid composition can nearly be matched and triglyceride
is biosynthesized with nearly the same proportions of SUS
as in cacao butter~
~ _ '
It will be understood that various changes and
~'~ modifications can be made in the details of procedure and
formulation without departing from the spirit of the in-
vention, especially as defined in the following claims.
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