Note: Descriptions are shown in the official language in which they were submitted.
7(~597
BACKGROUND OF THE :rNVENTION
Field of the invention
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The present invention relates to food industry
and, more specifically, to a method for optimization o~
microorganism cultures, particularly yeast cultures, spe-
cially lactic yeasts, for the production of biomass or of
intermediate fermentation metabolites, usable in food indus-
try.
Descri~tion of the ~rior art
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One of the known methods for cultivating lactic
yeasts on lactoserum is disclosed in French pàtent No.1,128,
063 of Fromageries Bel society. It relates to a method for
manufacturing burst lactic yeasts., which consists of sepa-
rating through precipitation nitrogenous fermentable bodies,
proteins and albumins, contained in the cheese-dairy or ca-
! ~ein-factory serum or in the serum-buttermilk mixture, and
of a~sorbing and converting by yeasts hydrocarbon bodies
exi~ting in the thus deproteinated serum or mixture. Fermen-
tation, run in precise pH, aeration and temperature condi-
tions, is carried out by Saccharomycés strains, lately
Glassified in the Kluyveromyces species (Lodder, 1970).
Since the filing date ~June 22, 1955) of French
patent No. 1,128,063, this method has been the subject matter
of many improvements, particularly as concerns the adaptation
of cultures to new techniques for deproteination of lacto-
serum, as well as conditioning and conservation of the thu~
obtained yeast cells. The deproteination of lactose, hereto-
fore carried out by thermal precipitation, is now realized by
ultrafiltration and ion exchange chromatography. Furthermore,
the carbon substrate, heretofore composed of lactose, is now
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composed of hydrolyzed lactose, such as indicated in French
patent application publication No. 2,439,819 filed by
Fromageries Bel society, wherein the culture of a micro-
organism is run on the milk substrate comprising hydrolyzed
lactose.
Presently, the production of food-yeast is
based on the preferential culture of some strains on some
media: for example, Kluyveromyces fragilis is cultivated
on lactoserum, Candida lipolytica on alkanes, Candida
tropicalis on gasoil and Candida utilis on molasses.
These fermentations, most often run in non
sterile conditions, result in the presence, in the mixture,
of other species, which, most often, are not desired.
Therefore, a resulting drawback is that the desired strain
can be brought to be eliminated from the fermentor by a
"contaminating pirate~ strain.
Moreover, if it is desired to obtain the
development of a pure culture, it is necessary that the
growth carbon substrate is of constant sterile quality
so as to maintain a single fermentation strain. Now,
lactoserums coming from the manufacture of lactic curd
cheeses or of lactic casein ~acidic lactoserums) contain 4
to 10 g/l of lactlc acid, whereas lactoserums coming from
the manufacture of rennet curd cheese or of rennet caseins
~mild lacto~erums) contain only 1 to 3 g/l of lactic
acid, Moreover, the change in the lactic acid content
of the substrate is also due to the fact that commercial
units for lactoserum treatment receive this material to be
treated continuously all day long and, as a function of
its origin, this lactoserum has a lactic acid ratio which
varies all day long.
In order to respond to the variation in this
ratio,
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it is necessary to devise a system able to adapt to these va-
riations by a selection of strains presenting a specificity
for assimilating substrates. Such a specificity, taking into
account ~e diauxy phenomena existin~ in a continuous culture,
can be considered only by the selection of mutants which will
have lost the capacity of assimilating the one or the other
one of the present carbon substrates. By the term "diauxy", it
should be understood the capacity of adaptation of a strain to
the consumption of various substrate~ the one~after the other
one, which imposes the selection between limited growth veloci-
ties so as to consume'all the carbon sources of fast growth
velocities and a loss of carbon substances.
The increase in the growth velocity of a culture
wherein there is a specificity of consumption of one species
with respect to a substrate is in fact translated by the satu-
ration of the'oxydative metabolism of Kluyveromyces and a por-
tion of the carbon source i5 downgraded by a fermentary way,
which leads to the compulsory appearance of ethanol. This etha-
j nol is known as a toxic metabolite for the cells; therefore,
its presence results in an inhibition of the yeast growth. Con-
sequently, there'is a lim$tation in the concentration of this
met'abolite'in ~his medium. It is therefore compulsory, in order
to impose maximum growth velocities, to remove this metabolite;
now, it represents a synthesis potentiality: A judicious way of
taking aavantage of this potentiality consists of introducing a
strain able'to assimilate'this substrate, in a specific way.
It would be therefore useful to cope with the draw-
backs of the usual method and to bring a solution to the herein-
before disclosed problems. An object of the present invention is
to allow the optimization of culture conditions on a lactic
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substrate, by using presently known culture techniques, and,
particularly, by using the opportunity of having mixed cultures
such as disclosed in an article of ~arrisson P. E. called "Mixed .
cultures in industrial fenmentation"'tAdvances in Applied
Mic~obiology (1978), 24, p. 129-164J which gives an example of
mixed cultures on methane and methanol. The use of mixed cul-
tures allows an increase in performances by using aifferent
substrates.
''SUMMARY'OF'THE'INVENTI'ON
An object of thé present'invention is to provide a
method for cultivation with an association of strains, each of
which will use a.different component of the medium, in an
exclusivé way, which provides an absence of competition with
re~pect to the''different other subst~ates of the medium.
Another object of the pre~s~n~ invention i~ to se-
lect specific mutants of a strain for a substrate in order to
create ecological "recesses", each "recess" corresponding to an
association of strai.ns pertaining to the ~ame species, strains
whi.ch will be'dependent upon the whole development of the cul-
ture,' the'refore upon neighbouring "reces'ses".
Other objects will appear from the following spe-
cification.
''DETAILED'DESCRIPTION'OF THE INVENTION
Thes'e ob~ects are now met by a method for cultiva-
ting microorganisms, particularly yeasts, on lacto~erum as a
starting substrate,' method wherein, after separation of pro-
teins contained in lactoserum and recovery of other components,
the culture is carried out on the thus obtained lactic substrate,
by using, on one hand, an association of judiciously selected
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strains, each of which is developing in a specific way on a
given substrate naturally present in the medium or on metabo-
lites'for~edduring the fermehtation process and, on the other
hand, specific mutants of a strain so as to create ecological
~recesses" corresponding to an association of strains pertai-
ning to the same species.
In the present invention, the culture media are
lactoserums coming from different milk and cheese treatments,
that is to say mild or acidic lactoserums, partially or wholly
deproteinatea by several known processes which can be used
commercially (precipitation t~rough thermal treatment, separa-
tion through ultrafiltration, exclusion and ion exchange chro-
matography, and so on) and'partially or wholly hydrolyzed
through an enzymatic way ttreatment with a ~ -galaotosidase) or
a chemical way.
Whatever the original culture medium may be, it
consists always, on one hand, of lactose (or a mixture of glucose,
galactose and lactose in the'case of a hydrolyzed lactose) and,
on the'other hand, of lactic acid and other o~ganic acids. The
proportion of both of these substrate categories can vary accor-
ding to the origin and the technology used for obtaining the
medium.
In the'method of the present in~ention, several
strains with high performances as concern~ their growth rate or
the protein yield of their composition and their specificity to
grow on a sub~trate'are selected, and they are associated with
a view to cultivate them on each of the carbon substrates pre-
sent even in a small amount in the starting natural material or
on metabolites formed during t~e ferme~tion ~ess. After s'ome
time,' a very stable flora balance is resulting, the proportion
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of the various species depending upon the concentration of
the substrates the ones with respect to the other ones.
In order to operate in an optimum way, such a
system requires ecological "recesses~. ~ithin each of them,
there exists a competition between various strains able to
grow with variable veIocities on the single present substrate.
It is ~bvious that the selec'tions within each of the "recesses"
are depending the onesupon the'other ones, particularly for
the "éthanol" recess, the substrate concentration of which is
depending upon the'fermentary activity of strains pertaining
to both of the other "recesses'l'tlactose and its hydrol~ysis
derivatives: lactic acid and other organic acids) and, par-
ticularly, to the ~lactose"' recess. '
The'selected strains pertain particularly to the
following species : KI~eromyces fragilis, Xluyveromyces lactis
and Torulopsi~ bovina. Kluyver'omyces fragilis are developing on
lactose, Kluyve~n~ces' lactis on lactic acid, without any possi-
bility of interferences, and Tbrulopsis bovina on ethanol. The
balance between strains is varying according to the proportion
of substrates'in the'medium and the 'capacity of the fermentor
for the'produced ethanol amount.
As an example, the'following distribution in a
medium consisting of lactose,' lactic acid and ethanol consisting
of 90-95% of lactoserum, 5-10% of lactic acid and 1-3~ of etha-
nol can be given :
- Kluyveromyces fragilis : about 90 to gS%
- Kluyveromyces lactis : about 5 to 10%
- Torulopsis bovina : about 1 to 3~
If this medium i~ partially or wholly hydrolyzed
lactoserum, which results in the presence of two additional
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carbon substrates, glucose ana galactose, it may be interes-
ting to use a new yeast strain such as Saccharomyces cerevisae
or Candida utilis. It should be noted that the hydrol~sis rate
of lactose can vary between 0 and 100~, the distribution of
glucose and galactose between 0 and 45% and that-of ~actosb-
hetween 0 and 95~.
The strain balance, which varies as a function of
the substrate nature,' is the following one, when there is
hydrolysis of lactose up to 80-90~ and the substrates are con-
sisiing of 10-20% of lactose, 36-43% of glucose, 36-43% of ga-
lactose, 5-10% of lactic acid and 1-3% of ethanol :
- Kluyveromyces fragilis : about 4~ to 65
- Kluyveromyces lactis : about 5 to 10 %
- Canaida utilis : about 35 to 45 %
- Torulopsis bovina : about 1 to 3 %
The used fermentors are operating, in optimum
conditions, at temperatures comprised between about 30 and 40C,
at a pJ~ of about 3 to 4, with an inlet air flow rate equal to
about 1300 m3~hr, with a range of about 2 to 3 billions of to-
tal cells per cm3, this rate being the same whatever the strain.It should be'noticed that the air volume being always a function
of the medium volume, the one of 1800 m3thr corresponds to a
medium volume of 25m3. By expressing the air volume not in m3thr
but in W m (air volume~medium volume~min~, the optimum condi-
tions are'located between 1.2 and 1.5. As a continuous work isc~rried out, the''flow rate is constant during the fermentation
and before tapping.
The biomass and~or metabolite 'yield can be increased
by incorporating within the'culture medium inorganic substances
such as phosphorus, copper, zinc, iron and many other elements
597
which are nutritive substances for microorganisms tH3Po
CuSO4,ZnSO4,FeSO4 addition).
- With respect to the presently existing culture
methods, with a single primordial strain and with parasitical
secondary strains, the method of the present invention has the
following advantages : ~
1 - It is possible to adapt the methoa to different
lactose~lactic acid ratios existing in lactoserums, which are
the culture natural occurring media.
2 - The whole'of each carbon source is used with a
limitation of lo~ses, whereby there is obtained an optimum
yield, which is about 50 to 55~. In the absence of ethanol me
tabolizing strain, the loss would have been at least equal to
the Torulopsis bovina proportion, i.e. 2 to 3%.
3 - It is possible to work with maximum growth ve-
locities of the main strains, due to the consumption by a third
~train of toxic intermediate metabolites, whereby there is ob-
talned a maximum productivity of the whole device ~fermentor);
the productivity gain i8 about 15 to 20% with respect to the
normal method with a single'primordial strain.
4 - The ~interrecess" rivalry (competition) is
cancel'led, while maintaining, on one'hand, rivalry inside each
rec'ess and, on the other hand, rivalry of strain~ between re-
cesses .
5 - The'plants are'not sophisticated as concerns
aeration and the"'limitation of air-liquid transLers which are
usually the 'factor limiting these'plants.
6 - The way of running operations is simplified.
The method of thé present invention allows the
production of biomass ~food-yeast) and/or intermediate metabolites.
117C~S97
The present invention will now be aisclosea with
the aia o~ the following examples which are given only as an
illustration ana not a limitation of the present invention.
EXAMPL~ 1 :
A mild lactoserum is used, coming from a cheese-
dairy, this lactoserum being deproteinatea by ultrafiltration.
This deproteinated lactoserum is sent to a so-callea
Airlift fermentor working in the following conditions :
- Useful volume : 23 m3
- - Flow rate : 7600 l/hr
- pH : 3 to 4
- Temperature - : 30 to 40C
- Air flow rate . : 1800 m3/hr
- Addition of a nitrogenous source : NH4OH
The selectea strains are the following ones, with the
hereinbelow indicated balance :
- Xluyveromyces fragilis on lactose : 90-96 ~
- ~luyveromyces lactis on lactic acid : 5-10 %
- Torulopsis bovina on ethanol : 1-3 %
Systematic controls, over two years, of the flora
present in the medium have shown a.very stable distribution
between the species.
~ EXAMPLE 2
An acidic lactoserum coming from a casein factory
i8 used; this lactoserum has been deproteinated by ultrafiltra-
tion and i.ts lactose ha~ been partially ~90%) hydrolyzed with
the aid of a p-galactosidase~
The fermentation i8 carried out according to the
same conditions as th.e ones given in example 1.
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The selected strains are the following o~es :
- Kluyveromyces fragilis on lactose and galactose
- Kluyveromyces lactis on lactic acid
- Candida utilis on glucose
- Torulopsis bovina on ethanol.
The distribution of the strains is the following
one :
- Kluyveromyces fragilis : 49.5 - 58.5 %
- Kluyveromyces lactis : 5 - 10 %
- Candidà utilis : 40 - 43 %
- Torulopsis bo~ina : 1 - 3 %
Systematic controls, over two years, of the flora
present in the medium have shown a very stable distribution
between the species.
Although the present specification relates to
preferred embodiments it should be understood that the invention
is not limited to said embodiments and include all modifications
and developments within the scope of the appended claims.
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