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Патент USA US3031400

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3,?3l,382
Patented Apr. 24, 1962
1
2
3,031,332
FERMENTATION PROCESS U?iNG SPORE
FORMENG FUNGI
Stanley G. Knight, Madison, Wis., assignor to Wisconsin
Alumni Research
Foundation, Madison, Wis., a cor
poratlon
of Wisconsin
No Drawing. Filed Sept. 15, 1958, Ser. No. ‘760,840
ing free fatty acids was converted to the desired highly
?avored composition much more rapidly by the old spore
7 Claims. (Cl. 195-101)
containing vegetative inoculum, in which the cells were
relatively inactive, than by the young substantially spore
free inoculum, in which cells were known to be relatively
active. With this unexpected discovery it was then de
cided to try the following experiment. Milk fat was
?rst modi?ed with lipase to release free fatty acids. Coni
dia or conidiospores of Penicillium roqueforti were then
The present invention relates to the microbiological 10 added to the modi?ed fat (free fatty acid) mixture and
production of chemical compounds by conidia or conidio
the mixture subjected to fermentation under submerged
spores (commonly called spores) produced during fer
mentation of a vegetative inoculum in an aqueous nu
aerobic conditions.
In a matter of minutes it was noted
that the spores (heretofore considered inert) were con
trient medium, and more speci?cally to the improvement
verting the free fatty acids to ketones. Instead of days,
which consists in the production of the compounds with 15 the desired ?avor was obtained in this manner in 24
the spores substantially free from vegetative inoculum in
hours.
a substantially nutrient-free aqueous medium. The
‘Investigations based on the above have shown that the
spores, unlike vegetative inoculum which is made up of
young active vegetative inoculum provides enzymes for
vegetative growth material, i.e. mycelium, are substan~
splitting the fat and that the free acids are then con
tially free from vegetative growth material. Also, un 20 verted to ketones when spores are produced in the old
like vegetative inoculum which requires a special fermen
relatively inactive vegetative inoculum. In this action
tation nutrient medium, the spores do not require a nu
the spores introduce a ketone (C=O) group on the num
trient medium.
ber 2 carbon atom and then decarboxylate the fatty acid.
Heretofore, microbiological production of chemical
compounds was carried outwith vegetative inoculum in a 25
fermentation medium containing nutrients such as corn~
This can be illustrated as follows:
spores
ll
steep liquor, soybean oil, soybean meal, molasses, sucrose,
Romolnomooon ——> RCHz-C-CHs
etc. to provide assimilable‘ sources of nitrogen and car
bon. See Fried et al. Patent 2,753,290‘ and Thoma et a1.
carbon atoms. Where R is butyl (C4H9) the reaction
where R is an alkyl group, e.g. containing 1--18 or more
Patent 2,793,462. The vegetative inoculum used was 30 can be illustrated as follows:
also made up of strong, young mycelium cells known to
be substantially spore-free but metabolically very active.
Recovery of the modi?ed chemicals from a medium con
taining vegetative growth material, nutrients, particularly
the complex organic nutrients, by-products of the same, 35
etc. obviously posed problems. Vegetative inoculum'
spores
OHaOH2CH2CHzCH2GH2CHiG O OH ——>
H
CH3CHtOH2CHzOHz—C-CH3
(Methyl, amyl ketone)
Free fatty acids can be used as such where available
and organic nutrients, however, were used as they were
considered’ essential in this art.
‘and where not available they can be made readily by
the fermentation period using vegetative inoculum (mold
not essential where the process is carried out with agita
tion or agitation accompanying aeration, the use of ho
mogenized fat such as in homogenized milk fat is pre
ferred both for the conversion of the fat to free fatty
acid by the enzymes and the conversion of the fatty acid
to the ketone by the spores. The overall action of the
enzymes and spores is materially speeded up when the
fat and fatty acid are dispersed in minute particles to pro-\
vide large areas of surface.
acid hydrolysis ‘of fats or by modi?cation of the corre
During my investigations of the conversion of fats, e.g.
sponding fats by use of fat splitting enzymes such as
milk fat, to highly ?avored compositions with molds of 40 lipase.
While homogenization of the fat or fatty acid is
the Penicillium type, e.g. P. roqueforti, it was noted that
cells‘ in mycelium form with nutrient growth medium)
was relatively short, e.g. 2 days, compared to the fer
mentation, e.g. 4 days, using mold conidia or could
io-spores. See copending Knight application, Serial No.
672,557, ?led July 18, 1957, now abandoned. It was also
noted that the fermentation period using e.g. 7 day old,
spore containing vegetative inoculum or spores alone was
longer than that required with young, eg 2 day old, sub
stantially spore-free vegetative inoculum. In view of
this it was assumed that the spores as such were rela
tively inert and that vegetative inoculum ‘and preferably
The spores used in the present invention can be readily
obtained from vegetative inoculum which hasbeen al
lowed to grow for 4-6 days in submerged culture with
aeration (e.g. shaking) in an aqueous cornsteep liquor
young inoculum should be used. Investigations also dis~
lactose or other nutrient medium in accordance with
closed that the fermentation mechanism apparently pro 55 standard
practices (except for culturing period) used to
ceeded ?rst by the hydrolysis or splitting of the fats and
prepare vegetative inoculum. The spores present in the
then by the conversion of the resulting free fatty acids
old vegetative inoculum are harvested by ?rst straining
to the desired highly ?avored aromatic ketones. It was
through cheesecloth to remove most of the mycelium and
also noted that the development of the desired ?avor
then by ?ltering through glass wool to remove the re
could be hastened by the addition of small amounts of 60 maining mycelium. The spores in the ?ltrate are re
lipase or like fat splitting enzyme. See Farnham Patent
covered by centrifuging and are then washed with water
2,531,329 and Knight application, supra. Up to this
to remove any retained nutrients. They may be stored in
time, however, it appeared that the use of vegetative inoc
‘dry form or resuspended in distilled water and be kept
ulurn with accompanying growth media (with or without
at 4° C. The suspension can be standardized to contain,
added‘ fat splitting enzymes) was necessary for short fer
for example, about one billion spores/ml. The spores
mentation periods, although it was appreciated that the
can also be grown on surface culture, e.g. nutrient agar,
use of spores was otherwise advantageous in that it was
‘and after scraping. off the spores and suspending in water,
easier, using a spore inoculum, to avoid or control un
the spores can be recovered in substantially pure form by
wanted secondary reactions caused by bacteria, etc.
70 ?ltering, centrifuging, etc., as described above.
In continued investigations it was noted, after lipase
The spore containing old vegetative inoculum can also
modi?ed the milk fat, that the resulting mixture contain
be used as such and, While this introduces vegetative
3,031,332
3
4
manner as the processes in the references including the
growth and some nutrients into the mixture, it represents
patents noted below, for example, except that the fer
a material improvement over the use of the nutrient
mentation of the vegetative inoculum in the nutrient me
dium, as used in the references and heretofore, is replaced
fermentation mixtures. When the vegetative inoculum is
used it can be recovered in fairly pure form free from
by the spores of the speci?ed organism, substantially free
from vegetative growth material, and the reaction is
soluble nutrients by centrifuging and by washing the re
covered solids with water. The use of spores, however,
carried out in a substantially nutrient-free aqueous me
substantially free from both vegetative growth and nutri
dium. If necessary, the aqueous mixtures should be
ents is preferred.
buffered
to provide a pH of about 6.5—7.5. The follow
The following examples are illustrative.
10 ing microbial modi?cations of steroids are illustrative:
Example I
(1) Reduction of progesterone to A4-pregnene-20/3-o1~
3-one with Streptomyces lavendulae. Fried, 1., et al., J.
About 10 mg. of lipase is ?rst added to about 1000 ml.
Am. Chem. Soc. 75:5764 (1953).
of homogenized milk containing added cream to provide
about 12% milk fat and the mixture shaken for about 4
hours at 37° C. The resulting fatty acid mixture is next 15
steamed for about 10 minutes to kill the lipase. After
cooling to 25° C., 200 ml. of an aqueous dispersion of
conidia or conidiospores of a white mutant of P. roque
(2) Dehydrogenation of secondary alcohols:
(a) A5-androstene-3?,17?-diol to testosterone with Pro
¢(zczin0myes erythropolis. Tur?tt, G. E., Biochem. J. 40:79
1946).
(b) Estradiol to estrone with Streptomyces albus.
Welsch, M., et al., Compt. rend. soc. biol, 142:1074
forti, prepared as described above, and containing about
a billion spores/m1., substantially free from vegetative 20 (1948).
(3) Hydroxylation in position 1: A4-androstene-3,17
inoculum and nutrients, are added to the fatty acid mix
dione to M-androstene-lu-ol-3,l7-dione with Penicillium
ture and the resulting mixture agitated with aeration at
sp., Dodson, R. M., et al., J. Am. Chem. Soc. 79:3921
about 1 vol/min. at a temperature of about 25° C. for
about 2.5 hours. The resulting highly ?avored composi
tion containing aromatic ketones derived from the fatty 25
acids can be concentrated and used as such or be obtained
(1957).
(4) Hydroxylation in position 2: A4-pregnene-l7a,21
diol-3,20-dione to A4-pregnene-2l8,17a,2l-triol-3,20-dione
with Streptomyces sp., Herzog, H. L., et al., J. Am. Chem.
in dry form by spray drying in accordance with estab
Soc. 7923922 (1957).
lished practices in the drying art. The white mutant used
(5) Hydroxylation in position 6: Progesterone to A4
in this example can be prepared as described in the refer
30 pregnene-6?-ol-3,20-dione with Streptomyces aureofaciens.
ences cited in the Knight application, supra.
Fried, 1., et al., Recent Progr. Hormone Res. 11:157
The 1 mg./ 100 ml. ratio of lipase to milk is merely
(1955). See also Dulaney, E. L., et al., Mycologia
illustrative as the amounts, depending on the percent of
47:464 (1955); Fried, J., et al., J. Am. Chem. Soc.
fat, temperature, time, etc. can vary widely with optimum
74:3962 (1952); Meister, P. D., et al., J. Am. Chem.
amounts for a particular composition being readily ascer
tained by preliminary test. The same applies to the 35 Soc. 75:416 (1953) and Eppstein, S. H., et al., I. Am.
Chem. Soc. 751408 (1953) involving the microorganisms
amount of spores used as the optimum amount (which
Aspergillus ochraceus, Aspergillus niger, and Rhizopus
can also be ascertained by preliminary test) can vary
arrhizus.
under different reaction conditions, different kind of
(6) Hydroxylation in position 7: Progesterone to A4
spores, etc.
Example II
40
pregnene-7a-ol-3,20-dione with Phycomyces blakeslecanus.
Fried et al., US. Patent 2,753,290. See also Meystre,
C., et al., Helv. Chim. Acta 38:381 (1955) using a spe
To a 1% aqueous mixture of octanoic acid bulfered
with phosphate buffer to pH 6.5 is added conidia or
cies of Peziza for a similar type reaction on deoxycorti
costerone.
conidio-spores of P. roqueforti, substantially free from
vegetative inoculum and nutrients, in a ratio of about 45
(7) Hydroxylation in positions 10, 11 or 12:
100,000,000 spores/3 ml. of the aqueous octanoic acid
mixture. The mixture is then subjected to agitation with
(a) l9-nor-progesterone to l0e-hydroxyl-l9'nonproges
terone with Rhizopus nigrlcans. Pederson, R. L., et al.,
aeration for about 2 hours at about 25° C. The result
J. Am. Chem. Soc. 7821512 (1956).
ing methyl, amyl ketone can be recovered by distillation
or like means known in the art.
Example III
This example follows Example II using hexanoic acid,
nonanoic acid and decanoic acid, respectively, for the
preparation of methyl, propyl ketone; methyl, hexyl ke
tone, and methyl, heptyl ketone. By selecting the ap
(b) Progesterone to A‘Lpregnene-lla-ol-3,20-dione with
50 Rhizopus arrhizus. Peterson, D. H., et al., I . Am. Chem.
Soc. 74:1871 (1952). See also Peterson, D. H., et al.,
J. Am. Chem‘. Soc. 752412 (1953).
(c) Reichstein’s compound S (A4-pregnene 17a,21-diol
3,20-dione) to hydrocortisone (A4-pregnene-ll?,l7a,2l
55
triol-3,20-dione with Cunninghamella blakesleeana. Han
son, F. R., et al., I. Am. Chem. Soc. 75:5369 (1953).
See also Shull, G. M., et al., I. Am. Chem. Soc. 772763
other ketones can be prepared in a similar manner with
(1955), and Thoma et al., US. Patent 2,793,162 involv
out vegetative inoculum or nutrients.
ing similar reactions ‘with Curvularia lunata, Trichotlze
60 cium roseum, and Coniothyrium hellebori.
Example IV
(d) Progesterone to A4-pregnene-12/3-ol-3,20-dione and
This example is in accordance with the above examples
the related 12?,15B-diol with Calonectria decora. Schu
propriate fatty acid containing at least 4 carbon atoms
but employs 4-—5 day old spore containing vegetative
bert, A., et al., Ber. 90, 2576 (1957).
(8). Hydroxylation in other positions are described in
soluble nutrients, dried and then ground to a powder and 65 Meister, P. D., et al., Absts. 123rd meeting Am. Chem.
resuspended in water. The ketones can be recovered by
Soc. (1953); Fried et al., US. Patent 2,753,290; Cam
distillation, extraction with water immiscible solvents, de
erino, B., et al., Gazz. Chim. ital., 86:1226 (1956);
cantation where the ketone is insoluble in water, etc.
Meystre, C., et al., Helv. Chim. Acta 38:381 (1955);
Separation of the ketones is not required when making a
Perlman, D., et al., J. Am. Chem. Soc. 74:2126 (1952);
?avoring composition as in Example I.
70 Thoma, R. W., et al., J. Am. Chem. Soc. 79:4818 (1957);
Dulaney, E. L., et al., Appl. Microbiol. 3:372 (1955);
In addition to converting fatty acids to ketones the im
Meystre, C., et al., Helv. Chim Acta 37:1548 (1954);
proved process of the present invention employing conidia
McAleer, W. J., et al., Arch. Biochem. Biophys. 62:109
or conidiospores can be used to advantage for the modi
?cation of other compounds including steroids or steroid
(1956).
inoculum which has been centrifuged to remove water
intermediates.
The process is carried out in the same 75
(9) Dehydrogenation: Hydrocortisone to A'-4-pregna
:H5M.
5
3,031,382
6
diene-l1,3,17a,21-triol-3,20-dione with Streptomyces la
tative growth material-free and nutrient-free aqueous me
dium in which the spores cannot germinate.
2. The process of claim 1 where the compounds are
vendulae. Fried et al., US. Patent 2,793,164. See also
Mobile, A., et al., J. Am. Chem. Soc. 77:4184 (1955);
Vischer, E., et al., Helv. Chim'. Acta 38:835 (1955).
(10) Side chain degradation: Progesterone to A'-4~
fatty acids.
3. In the microbiological production of a chemical
compound by fermentation of a fungi of the genus Strep
androstadiene-3,20-dione with Streptomyces lavendulae.
Peterson, G. E., et al., J. Bact. 742684 (1957). See also
Vischer, E., et al., Experienta 9:371 (1953); Tur?tt, G.
E., Biochem. I. 422376 (1948).
See also US. Patents 2,602,769; 2,649,400; 2,649,401;
2,649,402; 2,695,260; 2,735,800; 2,753,290; 2,762,747;
2,768,928; 2,789,940; 2,802,775; 2,809,919; 2,812,285;
tomyces, the improvement which consists in ?rst pro
ducing spores and vegetative growth material by cultur
ing the fungi in a nutrient medium, separating the spores
10 from the vegetative growth and nutrient materials, and
and 2,830,937 which involve microorganisms forming
spores which can be used in a nutrient-free medium in
place of the vegetative inoculum in the described proc
esses. Patents 2,602,769 and 2,735,800 listed above, use
fungus of the order Mucorales.
As noted above, the processes are carried out in ac
cordance with the processes described in the references
carrying out the production of the compound with the
spores in a substantially vegetative growth material-free
and nutrient-free aqueous medium in which the spores
cannot germinate.
4. In the microbiological production of a chemical
compound by fermentation of a fungi of the genus Peni
cillium, the improvement which consists in ?rst producing
spores and vegetative growth material by culturing the
fungi in a nutrient medium, separating the spores from
except (1) that the vegetative inoculum employed in the 20
the vegetative ‘growth and nutrient materials, and carry
reference process is replaced by spores of the organism
ing out the production of the compound with the spores
substantially free from vegetative growth material and
in a substantially vegetative growth material-free and nu
(2) that the aqueous nutrient fermentation medium
trient-free
aqueous medium in ‘which the spores cannot
employed in the reference process is replaced by a substan
germinate.
tially nutrient-free aqueous medium. The time required to 25
5. In the microbiological production of a chemical
complete the conversion with spores (and which can be
compound by fermentation of a fungi of the genus
readily ascertained by preliminary tests in each case)
Aspergillus, the improvement‘ which consists in ?rst
is materially shortened and the yield materially increased
producing spores and vegetative growth material by cul
over that using a young vegetative inoculum. Aside from
the fungi in a nutrient medium, separating the
the time element, however, the recovery of the modi?ed 30 turing
spores
from
the vegetative growth and nutrient materials,
chemical in ‘good yield (which, here again, can be car
and carrying out the production of the compound with
ried out in accordance with the reference process) is
the spores in a substantially vegetative growth material
much easier from the relatively clean reaction mixture
free and nutrient-free aqueous medium in which the
of the present invention than the prior art fermentation
cannot germinate.
mixtures containing vegetative growth, organic nutrients, 35 spores
6.
In
the microbiological production of a chemical
by-products of the same, etc. Puri?cation of the modi
compound by fermentation of a fungi of the genus
?ed compound after isolation is also much easier in the
Rhizopus, the improvement which consists in ?rst pro
process of the present invention than is possible from the
ducing spores and vegetative growth material by cultur
highly contaminated culture ?ltrates resulting from the
fermentation of vegetative inoculum in a nutrient me 40 ing the fungi in a nutrient medium, separating the spores
from“ the vegetative growth and nutrient materials, and
dium‘.
carrying out the production of the compound with the
As noted above lipase can be used to release free
spores in a substantially vegetative growth material-free
fatty acids from milk ‘fat. In the illustrative examples
and nutrient-free aqueous medium in which the spores
cannot germinate.
acids are converted to ketones. Whereas in these ex 45
7. In the microbiological production of a chemical
amples the reactions are conducted separately it should
compound by fermentation of a fungi of the order Mu
be understood that the enzymes and spores can be used
spores are added to the resulting mixture and free fatty
simultaneously.
corales, the improvement which consists in ?rst produc
ing spores and vegetative growth material by culturing
I claim:
1. In the microbiological production of a chemical com 50 the fungi in a nutrient medium, separating the spores
from the vegetative growth and nutrient materials, and
pound by fermentation of a spore forming fungi in an
carrying out the production of the compound with the
aqueous nutrient medium containing assimilable sources
spores in a substantially vegetative growth material-free
of nitrogen and carbon in which the spores can germi
and nutrient-free aqueous medium in which the spores
nate and grow and produce vegetative growth material,
the improvement which consists in ?rst producing the 55 cannot germinate.
spores by fermenting the spore forming fungi in said
nutrient medium, separating the spores from the result
ing fermentation medium containing vegetative growth
and nutrient materials and carrying out the production
of the compound with the spores in a substantially vege 60
References Cited in the ?le of this patent
Chem.
Peterson
Soc‘., et74,al.:5933-5936
DMS, March
(1952).
20, 1961, Iourn.
Dulaney et
Appl. MicrobioL, 32336-340 (1955).
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