Note: Descriptions are shown in the official language in which they were submitted.
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Method of separating lipids from a lysed lipids containing biomass
The current invention relates to a method of separating polyunsaturated fatty
acids containing lipids
from a lipids containing biomass.
PUFAs (polyunsaturated fatty acids) containing lipids are of high interest in
the feed, food and
pharmaceutical industry. Due to overfishing there is a high need for
alternative sources for PUFAs
containing lipids besides fish oil. It turned out that besides certain yeast
and algal strains in particular
microalgal cells like those of the order Thraustochytriales are a very good
source for PUFAs
containing lipids.
But with respect to microbial organisms and in particular cells of the order
Thraustochytriales, which
produce the PUFAs containing lipids, the isolation of the oil from the cells
turned out as a particular
problem. The most effective way of isolating the oil was the use of organic
solvents like hexane. But
the use of organic solvents leads to hazardous operating conditions, requires
the use of expensive
explosion-proof equipment and requires the implementation of an expensive
solvent recovery process
to avoid pollution of the environment.
.. In the attempt to avoid the use of organic solvents, as an effective
alternative way for isolating the oil
has turned out the salting-out of the oil with high amounts of sodium
chloride. But the use of high
amounts of sodium chloride leads to a delipidated biomass by-product which due
to the high salt
content cannot be utilized as a feed ingredient, so that the process is not
very sustainable. Further, the
high salt concentration leads to fast corrosion of the used steel equipment.
Thus, it was the object of the current invention to provide an effective
method for isolating a lipid, in
particular a PUFAs containing lipid, from lipids containing cells, in
particular of the order
Thraustochytriales, and simultaneously avoiding not only the need of organic
solvents, but further
avoid the need of high amounts of salts for realizing the effective isolation
of the oil from the cells.
It was a further object of the current invention to provide a method for
isolating a lipid, in particular a
PUFAs containing lipid, from lipids containing cells, in particular of the
order Thraustochytriales, and
simultaneously providing a delipidated biomass which can be utilized in a
commercial way, preferably
in the agricultural field.
It turned out that an effective separation of the lipid from the cell debris
containing aqueous phase can
be realized, if the lysed cell mixture is incubated at a low alkaline pH and a
temperature of not more
than 100 C for a time period of at least 10 hours. By keeping the temperature
below 100 C, it was
possible to prohibit at least essentially saponification of the fatty acid
esters.
Thus, a first subject of the current invention is a method of separating a
polyunsaturated fatty acids
(PUFAs) containing lipid from the debris of a biomass, comprising the
following steps:
a) Providing a suspension of a biomass comprising cells which contain a PUFAs
containing lipid;
b) Lysing the cells of the biomass;
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c) Heating the suspension as obtained in step (b) to a temperature of of 80
C to 100 C,
preferably 85 C to 95 C, more preferably about 90 C, while adjusting the pH to
a value of 9.5
to 11.5, preferably 10.0 to 11.0, more preferably 10.3 to 10.7;
d) Keeping the temperature and pH value in the ranges as depicted in (c) for
at least 10 hours,
preferably 15 to 40 hours, more preferably 20 to 36 hours.
The steps (c) and (d) lead to the separation of the oil containing light phase
and the water, cell debris,
salts and residual oil containing heavy phase, as obtained by lysing the cells
of the biomass. This
separation of the light and heavy phase is also called "de-emulsification" or
"demulsification" in the
context of this application.
The order of the measures in step (d) is of no importance. Adjusting of the
temperature can be carried
out before or after adjusting the pH value.
Preferably, in the steps (b), (c) and (d) of the method the suspension is
continuously mixed by using a
stirrer and/or an agitator. In the method steps (c) and/or (d) preferably low
shear agitation and/or axial-
flow agitation is applied, in particular as disclosed in WO 2015/095694.
Impellers suitable for agitating
prior and during steps (c) and/or (d) include in particular straight blade
impellers, Rushton blade
impellers, axial flow impellers, radial flow impellers, concave blade disc
impellers, high-efficiency
impellers, propellers, paddles, turbines and combinations thereof.
Lysing of the cells of the biomass can be carried out by methods as known to
those skilled in the art,
in particular enzymatically, mechanically, physically, or chemically, or by
applying combinations
thereof.
Depending on the time of exposure and/or the degree of force applied, a
composition comprising only
lysed cells or a composition comprising a mixture of cell debris and intact
cells may be obtained. The
term "lysed lipids containing biomass" insofar relates to a suspension which
contains water, cell debris
and oil as set free by the cells of the biomass, but beyond that may also
comprise further components,
in particular salts, intact cells, further contents of the lysed cells as well
as components of a
fermentation medium, in particular nutrients. In a preferred embodiment of the
invention, only small
amounts of intact cells, in particular less than 20 %, preferably less than 10
%, more preferably less
than 5 % (relating to the total number of intact cells as present before
lysing the cells of the biomass)
are present in the lysed biomass after the step of lysing the cells.
Lysing of the cells may be realized for example by utilizing a French cell
press, sonicator,
homogenizer, microfluidizer, ball mill, rod mill, pebble mill, bead mill, high
pressure grinding roll,
vertical shaft impactor, industrial blender, high shear mixer, paddle mixer,
and/or polytron
homogenizer.
In a preferred embodiment of the invention lysing of the cells is carried out
without applying high
mechanical stress on the cells. According to the invention, the energy input
onto the cells in the lysing
step preferably amounts to not more than 50 kWh per tonne of suspension, in
particular not more than
40, 30 or 20 kWh per tonne of suspension, especially preferably not more than
15, 10 or 5 kWh per
tonne of suspension.
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In a preferred embodiment of the invention, lysing of the cells comprises an
enzymatic treatment of the
cells by applying a cell-wall degrading enzyme.
According to the invention, the cell-wall degrading enzyme is preferably
selected from proteases,
cellulases (e.g., Cellustar CL (Dyadic), Fibrezyme G2000 (Dyadic), Celluclast
(Novozymes), Fungamyl
(Novozymes), Viscozyme L (Novozymes)), hemicellulases, chitinases, pectinases
(e.g., Pectinex
(Novozymes)), sucrases, maftases, lactases, alpha-glucosidases, beta-
glucosidases, amylases (e.g.,
Alphastar Plus (Dyadic); Termamyl (Novozymes)), lysozymes, neuraminidases,
galactosidases, alpha-
mannosidases, glucuronidases, hyaluronidases, pullulanases,
glucocerebrosidases,
galactosylceramidases, acetylgalactosaminidases, fucosidases, hexosaminidases,
iduronidases,
maltases-glucoamylases, xylanases (e.g., Xylanase Plus (Dyadic), Pentopan
(Novozymes)), beta-
glucanases (e.g., Vinoflow Max (Novozymes), Brewzyme LP (Dyadic)), mannanases,
and
combinations thereof. The protease may be selected from serine proteases,
threonine proteases,
cysteine proteases, aspartate proteases, metalloproteases, glutamic acid
proteases, alcalases
(subtilisins), and combinations thereof. The chitinase may be a
chitotriosidase. The pectinase may be
selected from pectolyases, pectozymes, polygalacturonases, and combinations
thereof.
The adequate pH for utilizing the enzyme depends on the pH optimum of the
enzyme. The pH
optimum of the enzyme is known to those skilled in the art or otherwise can be
determined easily.
In a preferred embodiment of the invention, an enzyme with a pH optimum of
between 6.5 and 8.5,
preferably of between 7.0 and 8.0, in particular of about 7.5, is used, so
that the pH applied in this step
is from 6.5 to 8.5, in particular from 7.0 to 8.0, preferably from 7.3 to 7.7.
A preferred enzyme which
can be used in this pH range is an alcalase.
The enzyme is preferably added as a concentrated enzyme solution, preferably
in an amount of 0.01
to 1.5 wt.-%, more preferably in an amount of 0.03 to 1.0 wt.-%, above all in
an amount of 0.05 to 0.5
wt.-%, relating to the amount of concentrated enzyme solution as added in
relation to the total amount
of the suspension after addition of the concentrated enzyme solution.
In a very preferred embodiment of the invention, lysing of the cells is
carried out as follows:
a) Heating the suspension of (a) to a temperature of between 50 C and 70 C,
preferably to a
temperature of between 55 C and 65 C, and adding a cell wall-degrading enzyme
to the
fermentation broth, and adjusting an adequate pH value, if necessary, at which
the enzyme is
properly working;
b) Keeping the temperature and pH in the ranges as depicted in (b) for at
least one hour,
preferably for at least two hours, more preferably for two to four hours.
In step (a), the enzyme can be added before or after heating up the suspension
and/or before or after
adjusting the pH. In the same way heating up of the suspension can be carried
out before or after
adjusting the pH. ¨ But in a preferred embodiment, the enzyme is added after
heating up of the
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suspension and after adjusting the pH, if adjusting of the pH is necessary, at
all. ¨ In a very preferred
embodiment all measures are carried out more or less simultaneously.
Preferably, in the steps (a) and (b) the suspension is continuously mixed by
using a stirrer and/or an
agitator.
In a further preferred embodiment of the invention, after lysing the cells of
the biomass and before the
demulsific,ation step, the suspension is concentrated to a total dry matter
content of 30 to 60 wt.-%,
more preferably 35 to 55 wt.-%, in particular 40 to 50 wt.-%.
Concentration of the suspension is preferably carried out by evaporation of
water at a temperature not
higher than 100 C, preferably 70 C to 100 C, more preferably 80 C to 90 C,
until a total dry matter
content of 30 to 60 wt.-% more preferably 35 to 55 wt.-%, in particular 40 to
50 wt.-%, is reached.
Concentration of the suspension is preferably carried out in a forced
circulation evaporator (for
example available from GEA, Germany) to allow fast removal of the water.
In general, adjusting the pH value can be carried out according to the
invention by using either bases
or acids as known to those skilled in the art. Decreasing of the pH can be
carried out in particular by
using organic or inorganic acids like sulfuric acid, nitric acid, phosphoric
acid, boric acid, hydrochloric
acid, hydrobromic acid, perchloric acid, hypochlorous acid, chlorous acid,
fluorosulfuric acid,
hexafluorophosphoric acid, acetic acid, citric acid, formic acid, or
combinations thereof. As a high
content of chloride is desirably avoided, in a preferred embodiment of the
invention no or only small
amounts of hydrochloric acid are used in the process of the current invention.
According to the
invention, sulfuric acid is the preferred substance for decreasing the pH
value. ¨ Increasing of the pH
can be carried out in particular by using organic or inorganic bases like
hydroxides, in particular
sodium hydroxide, lithium hydroxide, potassium hydroxide, and/or calcium
hydroxide, carbonates, in
particular sodium carbonate, potassium carbonate, or magnesium carbonate,
and/or bicarbonates, in
particular lithium bicarbonate, sodium bicarbonate, and/or potassium
bicarbonate. ¨ Due to easiness
of handling, the acids and bases are preferably used in liquid form, in
particular as concentrated
solutions. Thus, caustic soda is the preferred substance for increasing the pH
value.
The method according to the invention preferably comprises as a further step
the harvesting of the
PUFAs containing lipid from the demulsified composition as obtained in step
(d).
The harvesting of the PUFAs containing lipid preferably comprises
neutralization of the demulsified
suspension and subsequent separation of the thus obtained oil containing light
phase from the water,
salts, cell debris and residual oil containing heavy phase.
Neutralization of the demulsified composition is preferably realized by adding
an acid, preferably
sulfuric acid, to adjust a pH value of 5.5 to 8.5, in particular 6.5 to 8.5,
preferably 7.0 to 8Ø Before
starting separation of the light phase from the heavy phase the thus obtained
neutralized composition
may be stirred at said pH value from several minutes up to several hours.
Separation of the oil containing light phase from the water, salts and cell
debris containing heavy
phase is preferably realized by mechanical means and preferably at a
temperature of 60-90 C, more
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preferably 70-80 C, and at a pH value of preferably 6-9, more preferably 7-
8.5. "Mechanical means"
refers in particular to filtration and centrifugation methods as known to
those skilled in the art.
After separation of the oil containing light phase, the PUFAs containing oil
thus obtained can further be
worked up by applying methods as known to those skilled in the art, in
particular refining, bleaching,
deodorizing and/or winterizing.
A particular advantage of the method of the current invention is that it can
be carried out without the
use of any organic solvent, in particular without the use of any polar or non-
polar organic solvent.
Thus, in a preferred embodiment of the invention, no or only little amounts of
organic solvents, in
particular of polar or non-polar organic solvents, are used for isolating the
PUFAs containing oil from
the biomass. Typical organic solvents are hexane and ethanol.
In a preferred embodiment of the invention less than 2 wt.-% non-polar organic
solvents are used,
more preferably less than 1, 0.5 or 0.1 wt.-%. In a particularly preferred
embodiment of the invention
no non-polar organic solvent is used, at all. In a very preferred embodiment
of the invention less than
2 wt.-% organic solvents are used, in general, particularly preferred less
than 1, 0.5 or 0.1 wt.-%. In a
particularly very preferred embodiment of the invention no organic solvents
are used, at all, for
isolating the PUFAs containing oil from the biomass. ¨ This means in
particular for this embodiment
that the suspension as employed in the method according to the invention as
well as all compositions
as obtained by said single method steps preferably contain non-polar organic
solvents, preferably
organic solvents in general, in an amount of less than 2 wt.-%, more
preferably less than 1 wt.-%, in
particular less than 0.5 or 0.3 wt.-%, above all in an amount of less than 0.1
or 0.05 wt.-%.
A further advantage of the method of the current invention is that a very
effective separation of the oil
from the remaining biomass can be realized without the addition of sodium
chloride, which is normally
used for salting out the oil from the biomass. Preferably the method can be
carried out without the
addition of chloride salts, at all, above all without the addition of any
salts for salting out the oil. But
small amounts of chloride salts, in particular sodium chloride, might be
present in the suspension due
to the fermentation medium as used for growing of the biomass.
Thus, in a preferred embodiment of the current invention, no or only little
amounts of sodium chloride
are used for improving the oil isolation. In a preferred embodiment of the
invention less than 1 wt.-% of
sodium chloride, are used, more preferably less than 0.5 or 0.2 wt.-% of
sodium chloride are used for
isolating the oil from the biomass, above all less than 0.1 or 0.05 wt.-%,
wherein the wt.-% relate to the
total weight of the composition after addition of the sodium chloride. ¨ This
means in particular for this
embodiment that the suspension as employed in the method according to the
invention as well as all
compositions as obtained by said single method steps preferably contain sodium
chloride in an
amount of less than 2 wt.-%, more preferably less than 1 wt.-%, in particular
less than 0.5 or 0.3 wt.-
%, above all in an amount of less than 0.1 or 0.05 wt.-%.
In a particularly preferred embodiment of the invention no or only little
amounts of chloride salts are
used for improving the oil isolation, at all. In this embodiment preferably
less than 1 wt.-% of chloride
salts, more preferably less than 0.5 or 0.2 wt.-% of chloride salts are used
for isolating the oil from the
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biomass, above all less than 0.1 or 0.05 wt.-%, wherein the wt.-% relate to
the total weight of the
composition after addition of the chloride salts. ¨ This means in particular
for this embodiment that the
suspension as employed in the method according to the invention as well as all
compositions as
obtained by said single method steps preferably contain chloride, in
particular chloride salts, in an
amount of less than 2 wt.-%, more preferably less than 1 wt.-%, in particular
less than 0.5 or 0.3 wt.-
%, above all in an amount of less than 0.1 or 0.05 wt.-%.
In a very preferred embodiment of the invention no or only little amounts of
salts are used for
improving the oil isolation, in general. In this embodiment preferably less
than 1 wt.-% of salts, more
preferably less than 0.5 or 0.2 wt.-% of salts are used for isolating the oil
from the biomass, above all
less than 0.1 or 0.05 wt.-%, wherein the wt.-% relate to the total weight of
the composition after
addition of the salts. - This means in particular for this embodiment that the
suspension as employed
in the method according to the invention as well as all compositions as
obtained by said single method
steps preferably contain salts in general in an amount of less than 2 wt.-%,
more preferably less than
1 wt.-%, in particular less than 0.5 or 0.3 wt.-%, above all in an amount of
less than 0.1 or 0.05 wt.-%.
The methods of the current invention allow a very effective separation of the
oil contained in the
biomass from the cell-debris and other substances as contained in the
fermentation broth. By using
the methods of the current invention preferably more than 80 wt.-%, in
particular more than 90 wt.-%
of the oil contained in the biomass can be separated from the biomass and
isolated.
It turned out that the oil as obtained by applying the method of the current
invention has some
advantageous characteristics over the PUFAs containing oils as disclosed in
the state of the art so far.
In particular it exhibits very low oxidation values, a low content of free
fatty acids and impurities, a very
low viscosity and a very high flash point.
Thus, a further subject of the current invention is an oil as obtained or as
obtainable by a method
according to the current invention.
A further subject of the current invention is therefore also a PUFAs
containing oil exhibiting the
following characteristics: a) a peroxide value of less than 0.5, preferably
less than 0.3, in particular
less than 0.15; b) an anisidine value of less than 15, preferably less than
10; c) preferably a content of
free fatty acids of less than 1 wt.-%; d) preferably a content of moisture and
impurities of less than 1
wt.-%, preferably less than 0.5 wt.-%; e) preferably a viscosity of less than
250 cps, more preferably
of less than 200 cps, in particular of less than 160 cps; e) preferably a
flash point of at least 350 C,
more preferably of at least 400 C, in particular of at least 450 C; f)
preferably a content of omega-3
fatty acids, in particular of DHA and EPA, of at least 35 wt.-%, preferably at
least 40 or 45 wt.-%,
above all at least 50 wt.-%; g) preferably DHA and EPA each in an amount of at
least 8 wt.-%,
preferably at least 10 wt.-%, above all at least 15 wt.-%; h) preferably an
amount of organic solvents of
less than 0.5 wt.-%, more preferably less than 01 wt.-%, in particular less
than 0,05 wt.-%, above all
less than 0.01 wt.-%; i) preferably an amount of chlorides of less than 0.1
wt.-%, more preferably less
than 0.05 wt.-%, in particular less than 0.01 wt.-%; j) preferably a content
of crude fat of more than 90
wt.-%.
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The anisidine value (AV) is determined in accordance with AOCS Official Method
Cd 18-90. The AV is
a measure for secondary reaction products of the fatty acids, such as
aldehydes and ketones, that
occur during oxidation of the oil.
The peroxide value (PV) is determined in accordance with the AOCS Official
Method CD 8-53. The PV
is a measure for primary reaction products, such as peroxide and
hydroperoxides, that occur during
oxidation of the oil. ¨ According to the invention the PV is measured in
rrieq/kg.
The content of free fatty acids is determined in accordance with AOCS Official
Method AOCS Ca 5a-
40. The content of moisture is determined in accordance with AOCS Official
Methods AOAC 930.15,
935.29. The content of insoluble impurities is determined in accordance with
AOCS Official Method
AOCS 3a-46. The amount of DHA and EPA is determined in accordance with AOCS
Official Method
AOCS Ce lb-89. The amount of total fat is determined in accordance with AOCS
Official Method
AOCS 996.06. The amount of crude fat is determined in accordance with AOCS
Official Methods
AOAC 920.39, 954.02.
As the isolation of the oil is carried out by using no or only small amounts
of solvents and by also
using no or only small amounts of sodium chloride, the aqueous phase obtained
as a by-product is
preferably substantially free of organic solvents and sodium chloride, as
well. Thus, the aqueous
phase can be utilized in different ways, either directly after separation of
the oil phase or after further
work-up like concentrating and/or drying.
A further subject of the current invention is therefore a PUFAs containing
aqueous suspension,
containing a biomass, preferably a delipidated biomass, as obtained or as
obtainable by a method
according to the current invention. A further subject of the current invention
is therefore also a
concentrate or a dried product as obtained or obtainable by concentrating
and/or drying this aqueous
suspension. When concentrating the aqueous suspension, it is preferably dried
until a total dry matter
(TDM) content of 20 to 60 wt.-% is reached. ¨ In the following the expression
"aqueous suspension
according to the invention" refers to the aqueous phase as obtained after
separation of the oil phase
as well as to any concentrated suspensions of this aqueous phase as obtained
by concentrating of
this aqueous phase. Drying is preferably carried out by solvent evaporation,
as described further
below.
A further subject of the current invention is therefore also a PUFAs
containing aqueous suspension,
containing a biomass, in particular cell debris of a delipidated biomass,
characterized by a content of
non-polar organic solvents of less than 1 wt.-%, preferably less than 0.5 or
0.2 wt.-%, more preferably
less than 0.1 or 0.05 wt.-%, above all less than 0.01 wt.-%, and further
characterized by a content of
chloride ions of less than 1 wt.-%, preferably less than 0.5 or 0.2 wt.-%,
more preferably less than 0.1
or 0.05 wt.-%.
A further subject of the current invention is therefore in particular also a
PUFAs containing aqueous
suspension, containing a biomass, in particular, cell debris of a delipidated
biomass, characterized by
a content of organic solvents of less than 1 wt.-%, preferably less than 0.5
or 0.2 wt.-%, more
preferably less than 0.1 or 0.05 wt.-%, above all less than 0.01 wt.-%, and
further characterized by a
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content of chloride ions of less than 1 wt.-%, preferably less than 0.5 or 0.2
wt.-%, more preferably
less than 0.1 or 0.05 wt.-%.
A preferred subject of the current invention is therefore also a PUFAs
containing aqueous suspension,
containing a Thraustochytrid biomass, in particular cell debris of a
delipidated Thraustochytrid
biomass, characterized by a content of non-polar organic solvents of less than
1 wt.-%, preferably less
than 0.5 or 0.2 wt.-%, more preferably less than 0.1 or 0.05 wt.-%, above all
less than 0.01 wt.-%, and
further characterized by a content of chloride ions of less than 1 wt.-%,
preferably less than 0.5 or 0.2
wt.-%, more preferably less than 0.1 or 0.05 wt.-%.
A particularly preferred subject of the current invention is therefore also a
PUFAs containing aqueous
suspension, containing a Thraustochytrid biomass, in particular, cell debris
of a delipidated
Thraustochytrid biomass, characterized by a content of organic solvents of
less than 1 wt.-%,
preferably less than 0.5 or 0.2 wt.-%, more preferably less than 0.1 or 0.05
wt.-%, above all less than
0.01 wt.-%, and further characterized by a content of chloride ions of less
than 1 wt.-%, preferably less
than 0.5 or 0.2 wt.-%, more preferably less than 0.1 or 0.05 wt.-%.
The aqueous suspensions of the invention as described before preferably
exhibit a total dry matter
(TDM) content of 20 to 60 wt.-%, in particular of 25 to 55 wt.-%, more
preferably of 30 to 50 wt.-%, as
such concentrated suspensions turned out as particularly suitable for the
applications of the invention
as described below.
"Chloride" according to the invention refers to the amount of detectable
chlorine. The amount of
chlorine as present can be determined for example by elemental analysis
according to DIN EN ISO
11885. The chlorine is present in the form of salts which are called
"chlorides". The content of chloride
as mentioned according to the invention - also called "chloride ions" - only
refers to the amount of
detectable chlorine, not to the amount of the complete chloride salt, which
comprises besides the
chloride ion also a cationic counterion.
In a particularly preferred embodiment of the current invention the water,
salts, residual oil and cell
debris containing aqueous phase, which is obtained as by-product in the oil
harvesting step as
described before, is converted into a dried biomass by drying the biomass to a
total dry matter content
of more than 90 wt.-%.
Thus, a further subject of the current invention is also a PUFAs containing
biomass, in particular a
delipidated PUFAs containing biomass, characterized by a content of non-polar
organic solvents of
less than 2 wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%, more preferably
less than 0.1, 0.05 or 0.02
wt.-% and further characterized by a content of chloride ions of less than 2
wt.-%, preferably less than
1, 0.5 or 0.2 wt.-%, more preferably less than 0.1 or 0.05 wt.-%.
Thus, a further subject of the current invention is also a PUFAs containing
biomass, in particular a
delipidated PUFAs containing biomass, characterized by a content of organic
solvents of less than 2
wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%, more preferably less than
0.1, 0.05 or 0.02 wt.-% and
further characterized by a content of chloride ions of less than 2 wt.-%,
preferably less than 1, 0.5 or
0.2 wt.-%, more preferably less than 0.1 or 0.05 wt.-%.
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Thus, a preferred subject of the current invention is also a PUFAs containing
Thraustocyhtrid biomass,
in particular a delipidated Thraustochytrid biomass, characterized by a
content of non-polar organic
solvents of less than 2 wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%, more
preferably less than 0.1,
0.05 or 0.02 wt.-% and further characterized by a content of chloride ions of
less than 2 wt.-%,
preferably less than 1, 0.5 or 0.2 wt.-%, more preferably less than 0.1 or
0.05 wt.-%.
Thus, a particularly preferred subject of the current invention is also a
PUFAs containing
Thraustochytrid biomass, in particular a delipidated Thraustochytrid biomass,
characterized by a
content of organic solvents of less than 2 wt.-%, preferably less than 1, 0.5
or 0.2 wt.-%, more
preferably less than 0.1, 0.05 or 0.02 wt.-% and further characterized by a
content of chloride ions of
less than 2 wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%, more preferably
less than 0.1 or 0.05 wt.-%.
As preferably the preparation is carried out without the use of non-polar
organic solvents, preferably
without the use of any organic solvents, at all, and without the use of sodium
chloride, preferably
without the use of chloride salts, at all, the resulting biomass is preferably
free of any non-polar
organic solvents, preferably free of any organic solvents, in general, and
further essentially free of any
chloride ions, at all, wherein "essentially free" means that it contains
chloride ions in an amount of less
than 0.1 wt.-%, in particular in an amount of less than 0.05 wt.-%.
The biomass according to the invention exhibits preferably a moisture content
of not more than 10 wt.-
%, preferably not more than 5 wt.-%.
The biomass thus obtained preferably comprises lipids (crude fat) in an amount
of about 3 to 14 wt.-%,
in particular about 4 to about 14 wt.-%, preferably in an amount of about 4.5
to about 12 wt.-%, more
preferably in an amount of about 5 to about 10 wt.-%. Further, the lipid
preferably comprises at least
one PUFA selected from DHA and EPA, more preferably a mixture of DHA and EPA,
wherein the ratio
of DHA to EPA is preferably between 3:2 to 4:1 and wherein the amount of DHA
is preferably from 30
to 50 wt.-% of the total amount of lipids contained and the amount of EPA is
preferably from 10 to 20
wt.-%. of the total amount of lipids contained. Accordingly, also the aqueous
suspensions as described
before are preferably characterized by being convertible by drying into a
biomass with such a crude fat
content and/or EPA content and/or DHA content by drying the aqueous suspension
to a moisture
content of not more than 10 wt.-%, preferably not more than 5 wt.-%.
The biomass preferably further comprises amino acids in an amount of 15 to 25
wt.-%, more
preferably in an amount of 17 to 23 wt.-%, and exhibits preferably a crude
protein content of 25 to 35
wt.-%. Accordingly, also the aqueous suspensions as described before are
preferably characterized
by being convertible by drying into a biomass with such an amino acid and/or
crude protein content by
drying the aqueous suspension to a moisture content of not more than 10 wt.-%,
preferably not more
than 5 wt.-%.
.. The biomass preferably further exhibits a crude fiber content of less than
5 wt.-%, preferably less than
2 wt.-%, more preferably of about 0 wt.-%. Accordingly, also the aqueous
suspensions as described
before are preferably characterized by being convertible by drying into a
biomass with such a crude
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fiber content by drying the aqueous suspension to a moisture content of not
more than 10 wt.-%,
preferably not more than 5 wt.-%.
The dried biomass is preferably a delipidated biomass, that means a biomass,
of which the major part
of the lipids have been removed, preferably by a process as disclosed in this
application. As the
separation of oil from the biomass is very effectively, the remaining oil in
the biomass is preferably less
than 20 wt.-%, preferably less than 15 wt.-%, more preferably less than 10 wt.-
%, of the oil as
originally contained in the biomass. But as the oil cannot be removed
completely by such a process, a
substantial amount of oil is still contained also in the delipidated biomass
according to the invention.
That means that the term "delipidated biomass" according to the invention
refers to a lysed biomass,
from which the major part of oil has been removed, preferably by a process or
method as disclosed in
this application, but which still contains a substantial part of lipids, in
particular of PUFAs containing
lipids, wherein the amount of lipids in the dried delipidated biomass is
preferably from 3-14 wt.-%, in
particular 4-14 wt.-%, preferably from 4.5-12 wt.-%, more preferably from 5-10
wt.-%. Thus, the
"delipidated biomass" according to the invention might also be called a
"partially delipidated biomass"
or a "substantially delipidated biomass".
Thus, a further subject of the current invention is a method of obtaining a
biomass which is
substantially free of non-polar organic solvents, preferably free of organic
solvents, in general, and
which is further substantially free of sodium chloride, preferably free of
chloride salts, in general,
comprising the method steps as mentioned before.
Conversion of the water, salts, remaining oil and cell debris containing heavy
phase, which is obtained
as by-product in the oil harvesting step, into a dried biomass by drying the
biomass to a total dry
matter content of more than 90 wt.-%, can be carried out in different ways.
In a very preferred way, the transformation is carried out by concentration of
the heavy phase to a dry
matter content of 30-50 wt.-%, preferably 35-45 wt.-%, and subsequent spray
granulation of the
biomass by means of fluidized bed granulation. By doing that, in a very
efficient way, a biomass with
advantageous features can be obtained. Spray granulation by means of fluidized
bed granulation is
disclosed in more detail in EP13176661Ø
The biomass as obtained in that way has some further advantageous
characteristics as follows: it has
a good flowability (preferably at least grade 4), a low dust value (preferably
free of dust), a high bulk
density of preferably more than 500 kg/m3, and/or a high energy value of at
least 3500 kcal/kg,
preferably of about 3800 to 4200 kcal/kg.
Concentration of the heavy phase to a dry matter content of 30-50 wt.-% is
preferably carried out by
solvent evaporation, in particular vacuum evaporation, and/or by using a
rotary evaporator, a thin-film
evaporator or a falling-film evaporator. A useful alternative to solvent
evaporation is reverse osmosis.
For determining the flowability of the granular biomass conical glass efflux
vessels with different size
outflow openings are used (Klein: Seifen, Ole, Fette, Wachse 94, 12 (1968)).
The glass vessels exhibit
a height of 70 mm, a maximal inner diameter of 36 mm, a maximal outer diameter
of 40 mm and
circular apertures at the conical end of the glass vessels with diameters as
follows: 2.5; 5; 8; 12; 18
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mm. The glass vessels are completely filled with the granular biomass and
subsequently fixed in a
rack with the aperture directed downwards. Preferably, the aperture of the
glass vessels is opened by
removing a covering located on the aperture after having fixed the glass
vessels on the rack.
The flowability is determined as follows: If the granular material can flow
out of the vessel with the
smallest diameter (2.5 mm) without stagnation, then the flowability is
determined as 1; if it can flow out
of the vessel with diameter of 5 mm without stagnation, then the flowability
is determined as 2; and so
on. A flowability of 6 means that the granular material can not flow out of
the vessel with the broadest
diameter (18 mm), at all, or it can flow out of this vessel only with
stagnation. ¨ Thus, a flowability of 4
means that the granular material can flow out of the vessel with a diameter of
12 mm without
stagnation.
"Dust-free" according to the invention is understood to mean a powder which
contains only low
fractions (< 10% by weight, preferably < 5% by weight, in particular < 3% by
weight, especially < 1%
by weight) of particle sizes below 100 micrometres.
In a preferred embodiment of the invention, a fraction of at least 80% by
weight, in particular at least
90% by weight, particularly preferably at least 95% by weight, especially at
least 98% by weight of the
particles of the biomass possess a particle size of from 100 to 2500
micrometres, preferably 300 to
2500 micrometres, in particular 500 to 2200 micrometres, more preferably 1000
to 2000 micrometers.
The mean particle diameter d50 of the particles of the biomass is preferably
in the range of 500 to
2200 micrometers, more preferably in the range of 1000 to 2000 micrometers, in
particular in the
range of 1300 to 1900 micrometers.
Grain or particle size is preferably determined according to the invention by
laser diffraction
spectrometric methods. Possible methods are described in the text book
"Teilchengrogenmessung in
der Laborpraxis" [Particle size measurement in the laboratory] by R. H. Muller
arid R. Schuhrnann,
Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) and in the text book
"Introduction to Particle
Technology" by M. Rhodes, Wiley & Sons (1998). Inasmuch as various methods can
be used, the
first-cited usable method from the text book of R.N. Mailer and R. Schuhmann
for the measuring of
particle size is preferably used.
The bulk density of the biomass according to the invention is preferably 400
to 800 kg/m3, particularly
preferably 450 to 750 kg/m3, in particular 500 to 750 kg/m3.
As alternative to the spray-granulation other drying methods, in particular
other convective drying
methods, like tunnel drying or spray drying, in particular nozzle spray
drying, or contact drying
methods, like drum drying, or radiation drying methods, like infrared drying,
of the concentrated heavy
phase would be applicable alternatives, wherein by using those methods
normally particles with a
smaller or bigger diameter are obtained.
According to the invention, during the drying process, an anti-caking agent,
in particular silica,
preferably a hydrophobic or hydrophilic silica, may optionally be added to the
biomass to prevent
caking. For this purpose, the fermentation broth comprising biomass as well as
the silica are
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preferably sprayed into the particular drying zone. Alternatively or
additionally, the biomass may be
mixed with the anti-caking agent after the drying process. With respect to the
use of silica as anti-
caking agent reference is made in particular to the patent application
EP13187631Ø
In a particular embodiment, the biomass according to the invention has a
concentration of an anti-
caking agent, in particular silica, preferably hydrophilic or hydrophobic
silica, of 0.2 to 10% by weight,
in particular 0.5 to 7% by weight, especially 0.5 to 5% by weight.
Conversion of a fine-grained powder into a coarse-grained dust-free product
can be realized by
granulating processes. Conventional organic or inorganic auxiliaries or
supports such as starch,
gelatin, cellulose derivatives or similar substances, which are typically used
in food processing or feed
processing as binding agents, gelling agents or thickeners, may optionally be
used in this subsequent
granulation process. Further auxiliaries that are preferably used according to
the invention are
disclosed in WO 2016/050560, with carboxymethylcellulose being a particulary
preferred binding
agent.
Thus, in a particular embodiment of the current invention, the biomass
contains an agglomeration
auxiliary, in particular a modified polysaccharide, preferably
carboxymethylcellu lose, in an amount of
from 0.05 to 10 wt.-%, preferably in an amount of 0.1 to 5 wt.-%.
A product having the desired particle size and/or particle size distribution
can optionally be obtained
from the granulate as obtained by drying and/or granulation by subsequent
sieving or dust separation.
After drying and optionally granulating and/or sieving of the biomass, the
dried biomass is preferably
stored or packed.
The particulate biomass of the invention as well as the aqueous suspensions of
the invention can be
used in different ways. For example, they can be used in order to produce a
foodstuff or feedstuff, as
the biomass and aqueous suspensions according to the invention surprisingly
turned out to be well
accepted as feed ingredient by animals, in particular by beef cattle.
Alternatively they may be used
directly as foodstuff or feedstuff.
A feedstuff or foodstuff comprising a particulate biomass or an aqueous
suspension according to the
invention is therefore a further subject matter of the present invention. The
feedstuff may for example
be used for feeding poultry, swine, minks, ruminants, in particular beef
cattle or calves, sheep, goats,
companion animals or animals hold in aquaculture. In a very preferred
embodiment of the invention,
the feedstuff is used for feeding beef cattle. The feedstuff or foodstuff
preferably comprises the
biomass in an amount of 2 to 60 wt.-%, preferably in an amount of 5 to 50 wt.-
%, more preferably in an
amount of 10 to 30 wt.-%.
A further subject matter of the present invention is therefore likewise the
use of a particulate biomass
and/or of an aqueous suspension according to the invention for producing a
foodstuff or feedstuff.
A further subject matter of the present invention is therefore likewise a
method for producing a
feedstuff or foodstuff, in which a particulate biomass and/or an aqueous
suspension according to the
invention is used, and is preferably mixed with further feedstuff or foodstuff
ingredients.
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In a preferred embodiment of the invention, the particulate biomass and/or the
aqueous suspension is
used for producing a foodstuff or feedstuff, in which the biomass and/or the
aqueous suspension is
preferably mixed with other foodstuff or feedstuff ingredients and is then
processed to give the
foodstuff or feedstuff.
The mixture of biomass and/or aqueous suspension and other foodstuff or
feedstuff ingredients is
processed in a preferred embodiment by an extrusion process, in order to
obtain portions of foodstuff
or feedstuff ready for sale. Alternatively, a pelleting method may also be
used.
A screw or twin-screw extruder is preferably employed in the extrusion
process. The extrusion process
is preferably carried out at a temperature of 80¨ 220 C, particularly 100¨ 190
C, a pressure of 10 ¨
40 Bar, and a shaft rotational speed of 100¨ 1000 rpm, particularly 300 ¨700
rpm. The residence
time of the mixture introduced is preferably 5-30 seconds, in particular 10 ¨
20 seconds.
In a mode of the extrusion process which is preferred in accordance with the
invention, the process
comprises a compacting step and a compression step.
It is preferred to intimately mix the components with each other before
carrying out the extrusion
process. This is preferably carried out in a drum equipped with vanes. In this
mixing step, a preferred
embodiment includes an injection of steam, in particular so as to bring about
the swelling of the starch
which is preferably present.
Before being mixed with the biomass and/or aqueous suspension, the further
foodstuff or feedstuff
ingredients are preferably comminuted ¨ if required ¨ so as to ensure that a
homogeneous mixture is
obtained in the mixing step. The comminuting of the further foodstuff or
feedstuff ingredients may be
carried out, for example, using a hammer mill.
A further subject of the current invention is therefore a method of feeding
animals, wherein a
particulate biomass and/or an aqueous suspension according to the invention
are provided to animals,
preferably after mixing the particulate biomass and/or the aqueous suspension
with further feedstuff
ingredients, wherein the animals are preferably selected from poultry, swine,
minks, ruminants, in
particular from calves and beef cattle, sheep, goats, companion animals or
animals hold in
aquaculture.
Alternatively the biomass and/or aqueous suspension according to the invention
may be used in land
applications, in particular as (organic) fertilizer, NPC
(nitrogen/phosphorous/potassium source), soil
enhancer, plant enhancer and/or composting aid, for producing biogas, for
wastewater treatment or as
alternative fuel, in particular for cement kilns. It might be further used as
part of a fermentation medium
for producing microorganisms, in particular for producing further PUFAs
containing biomass.
A further subject of the current invention is therefore a method for enhancing
soil, wherein a
particulate biomass and/or an aqueous suspension according to the invention
are strewed on and
possibly mixed with ground, in particular with farmland soil or garden soil.
A further subject of the current invention is therefore also a method for
fertilizing and/or composting
ground, in particular farmland or garden, wherein a particulate biomass and/or
an aqueous suspension
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according to the invention are strewed on and possibly mixed with ground, in
particular with farmland
soil or garden soil.
A further subject of the current invention is therefore also a method for
producing biogas, wherein a
particulate biomass and/or an aqueous suspension according to the invention is
subjected to microbial
degradation under anaerobic conditions, in particular by making use of
methanogenic bacteria.
A further subject of the current invention is therefore also a method for
treatment of wastewater,
wherein wastewater is mixed with a particulate biomass and/or an aqueous
suspension according to
the invention.
A further subject of the current invention is therefore also a method for
producing microorganisms, in
particular for producing a PUFAs containing biomass, wherein a particulate
biomass and/or aqueous
suspension according to the invention is used as part of the fermentation
medium.
The method according to the invention may further comprise as a pretreatment
step the pasteurization
of the suspension of the biomass, before carrying out the lysis of the cells.
The pasteurization is
preferably carried out for 5 to 80 minutes, in particular 20 to 60 minutes, at
a temperature of 50 to
121 C, in particular 50 to 70 C.
The PUFAs containing cells of the biomass are preferably microbial cells or
plant cells. Preferably, the
cells are capable of producing the PUFAs due to a polyketide synthase system.
The polyketide
synthase system may be an endogenous one or, due to genetic engineering, an
exogenous one.
Accordingly, "delipidated biomass" according to the invention in particular
refers to the residues of
such a PUFAs containing cells comprising biomass, in particular as disclosed
further below, after
having been subjected to an oil isolation process, in particular as disclosed
further before.
The plant cells may in particular be selected from cells of the families
Brassicaceae, Elaeagnaceae
and Fabaceae. The cells of the family Brassicaceae may be selected from the
genus Brassica, in
particular from oilseed rape, turnip rape and Indian mustard; the cells of the
family Elaeagnaceae may
be selected from the genus Elaeagnus, in particular from the species Oleae
europaea; the cells of the
family Fabaceae may be selected from the genus Glycine, in particular from the
species Glycine max.
The microbial organisms which contain a PUFAs containing lipid are described
extensively in the prior
art. The cells used may, in this context, in particular be cells which already
naturally produce PUFAs
(polyunsaturated fatty acids); however, they may also be cells which, as the
result of suitable genetic
engineering methods or due to random mutagenesis, show an improved production
of PUFAs or have
been made capable of producing PUFAs, at all. The production of the PUFAs may
be auxotrophic,
mixotrophic or heterotrophic.
The biomass preferably comprises cells which produce PUFAs heterotrophically.
The cells according
to the invention are preferably selected from algae, fungi, particularly
yeasts, bacteria, or protists. The
cells are more preferably microbial algae or fungi.
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Suitable cells of oil-producing yeasts are, in particular, strains of
Yarrowia, Candida, Rhodotorula,
Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
Suitable cells of oil-producing nnicroalgae and algae-like microorganisms are,
in particular,
microorganisms selected from the phylum Stramenopiles (also called
Heterokonta). The
microorganisms of the phylum Stramenopiles may in particular be selected from
the following groups
of microorganisms: Hannatores, Proteromonads, Opalines, Developayella,
Diplophrys, Labrinthulids,
Thraustochytrids, Biosecids, Oomycetes, Hypochytridionnycetes, Commation,
Reticulosphaera,
Pelagomonas, Pelagococcus, 011icola, Aureococcus, Parmales, Diatoms,
Xanthophytes, Phaeophytes
(brown algae), Eustigmatophytes, Raphidophytes, Synurids, Axodines (including
Rhizochronnulinales,
Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales,
Hibberdiales, and
Chromulinales. Other preferred groups of microalgae include the members of the
green algae and
dinoflagellates, including members of the genus Crypthecodiurn.
The biomass according to the invention preferably comprises cells, and
preferably consists essentially
of such cells, of the taxon Labyrinthulomycetes (Labyrinthulea, net slime
fungi, slime nets), in
particular those from the family of Thraustochytriaceae. The family of the
Thraustochytriaceae
(Thraustochytrids) includes the genera Althomia, Aplanochytrium,
Aurantiochytrium, Botryochytrium,
Elnia, Japonochytrium, Oblong ichytrium, Parietichytrium, Schizochytrium,
Sicyoidochytrium,
Thraustochytrium, and Ulkenia. The biomass particularly preferably comprises
cells from the genera
Aurantiochytrium, Oblongichytrium, Schizochytrium, or Thraustochytriunn, above
all from the genus
Schizochytrium.
In accordance with the invention, the polyunsaturated fatty acid (PUFA) is
preferably a highly-
unsaturated fatty acid (HUFA).
The cells present in the biomass are preferably distinguished by the fact that
they contain at least 20%
by weight, preferably at least 30% by weight, in particular at least 35% by
weight, of PUFAs, in each
case based on cell dry matter.
According to the current invention, the term "lipid" includes phospholipids;
free fatty acids; esters of
fatty acids; triacylglycerols; sterols and sterol esters; carotenoids;
xanthophylls (e. g., oxycarotenoids);
hydrocarbons; isoprenoid-derived compounds and other lipids known to one of
ordinary skill in the
art. ¨ The terms "lipid" and "oil" are used interchangeably according to the
invention.
In a preferred embodiment, the majority of the lipids in this case is present
in the form of triglycerides,
with preferably at least 50% by weight, in particular at least 75% by weight
and, in an especially
preferred embodiment, at least 90% by weight of the lipids present in the cell
being present in the form
of triglycerides.
According to the invention, polyunsaturated fatty acids (PUFAs) are understood
to mean fatty acids
having at least two, particularly at least three, C-C double bonds. According
to the invention, highly-
unsaturated fatty acids (HUFAs) are preferred among the PUFAs. According to
the invention, HUFAs
are understood to mean fatty acids having at least four C-C double bonds.
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The PUFAs may be present in the cell in free form or in bound form. Examples
of the presence in
bound form are phospholipids and esters of the PUFAs, in particular monoacyl-,
diacyl- and
triacylglycerides. In a preferred embodiment, the majority of the PUFAs is
present in the form of
triglycerides, with preferably at least 50% by weight, in particular at least
75% by weight and, in an
especially preferred embodiment, at least 90% by weight of the PUFAs present
in the cell being
present in the form of triglycerides.
Preferred PUFAs are omega-3 fatty acids and omega-6 fatty acids, with omega-3
fatty acids being
especially preferred. Preferred omega-3 fatty acids here are the
eicosapentaenoic acid (EPA, 20:5w-
3), particularly the (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid,
and the
docosahexaenoic acid (DHA, 22:6w-3), particularly the (4Z,7Z,10Z,13Z,16Z,19Z)-
docosa-
4,7,10,13,16,19-hexaenoic acid.
In a very preferred embodiment of the current invention, cells, in particular
a Schizochytrium strain, is
employed which produces a significant amount of EPA and DHA, simultaneously,
wherein DHA is
preferably produced in an amount of at least 20 wt.-%, preferably in an amount
of at least 30 wt.-%, in
.. particular in an amount of 30 to 50 wt.-%, and EPA is produced in an amount
of at least 5 wt.-%,
preferably in an amount of at least 10 wt.-%, in particular in an amount of 10
to 20 wt.-% (in relation to
the total amount of lipid as contained in the cells, respectively). DHA and
EPA producing
Schizochytrium strains can be obtained by consecutive mutagenesis followed by
suitable selection of
mutant strains which demonstrate superior EPA and DHA production and a
specific EPA:DHA ratio.
Any chemical or nonchemical (e.g. ultraviolet (UV) radiation) agent capable of
inducing genetic
change to the yeast cell can be used as the mutagen. These agents can be used
alone or in
combination with one another, and the chemical agents can be used neat or with
a solvent.
Preferred species of microorganisms of the genus Schizochytrium, which produce
EPA and DHA
simultaneously in significant amounts, as mentioned before, are deposited
under ATCC Accession No.
PTA-10208, PTA-10209, PTA-10210, or PTA-10211, PTA-10212, PTA-10213, PTA-
10214, PTA-
10215.
The suspension of biomass according to the present invention, from which the
PUFAs containing lipid
is obtainable, is preferably a fermentation broth, in particular a
fermentation broth with a biomass
density of at least 80 or 100 g/I, preferably at least 120 or 140 g/I, more
preferably at least 160 or 180
g/I (calculated as dry-matter content). Thus, the suspension may be obtained
by culturing and growing
suitable cells in a fermentation medium under conditions whereby the PUFAs are
produced by the
microorganism.
Methods for producing the biomass, in particular a biomass which comprises
cells containing lipids, in
particular PUFAs, particularly of the order Thraustochytriales, are described
in detail in the prior art
(see e.g. W091/07498, W094/08467, W097/37032, W097/36996, W001/54510). As a
rule, the
production takes place by cells being cultured in a fermenter in the presence
of a carbon source and
of a nitrogen source, along with a number of additional substances like
minerals that allow growth of
the microorganisms and production of the PUFAs. In this context, biomass
densities of more than 100
grams per litre and production rates of more than 0.5 gram of lipid per litre
per hour may be attained.
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The process is preferably carried out in what is known as a fed-batch process,
i.e. the carbon and
nitrogen sources are fed in incrementally during the fermentation. When the
desired biomass has
been obtained, lipid production may be induced by various measures, for
example by limiting the
nitrogen source, the carbon source or the oxygen content or combinations of
these.
.. In a preferred embodiment of the current invention, the cells are grown
until they reach a biomass
density of at least 80 or 100 g/I, more preferably at least 120 or 140 g/I, in
particular at least 160 or
180 g/I (calculated as dry-matter content). Such processes are for example
disclosed in US 7,732,170.
Preferably, the cells are fermented in a medium with low salinity, in
particular so as to avoid corrosion.
This can be achieved by using chlorine-free sodium salts as the sodium source
instead of sodium
chloride, such as, for example, sodium sulphate, sodium carbonate, sodium
hydrogen carbonate or
soda ash. Preferably, chloride is used in the fermentation in amounts of less
than 3 g/I, in particular
less than 500 mg/I, especially preferably less than 100 mg/I.
Suitable carbon sources are both alcoholic and non-alcoholic carbon sources.
Examples of alcoholic
carbon sources are methanol, ethanol and isopropanol. Examples of non-
alcoholic carbon sources are
fructose, glucose, sucrose, molasses, starch and corn syrup.
Suitable nitrogen sources are both inorganic and organic nitrogen sources.
Examples of inorganic
nitrogen sources are nitrates and ammonium salts, in particular ammonium
sulphate and ammonium
hydroxide. Examples of organic nitrogen sources are amino acids, in particular
glutamate, and urea.
In addition, inorganic or organic phosphorus compounds and/or known growth-
stimulating substances
such as, for example, yeast extract or corn steep liquor, may also be added so
as to have a positive
effect on the fermentation.
The cells are preferably fermented at a pH of 3 to 11, in particular 4 to 10,
and preferably at a
temperature of at least 20 C, in particular 20 to 40 C, especially preferably
at least 30 C. A typical
fermentation process takes up to approximately 100 hours.
After the fermentation has ended, the cells may be pasteurized in order to
kill the cells and to
deactivate enzymes which might promote lipid degradation. The pasteurization
is preferably effected
by heating the biomass to a temperature of 50 to 121 C, preferably 50 to 70 C,
for a period of 5 to 80
minutes, in particular 20 to 60 minutes.
Likewise, after the fermentation is ended, antioxidants may be added in order
to protect the PUFAs
present in the biomass from oxidative degradation. Preferred antioxidants in
this context are BHT,
BHA, TBHA, ethoxyquin, beta-carotene, vitamin E, in particular tocopherol, and
vitamin C. The
antioxidant, if used, is preferably added in an amount of 0.001 to 0.1 wt.-%,
preferably in an amount of
0.002 to 0.05 wt.-%, relating to the total amount of the fermentation broth
after addition of the
antioxidant.
18
Working examples
Example 1
An unwashed cell broth containing microbial cells (Schizochytrium sp.) at a
biomass density of over
100 g/I was heated to 60 C in an agitated vessel. After heating up the
suspension, the pH was
adjusted to 7.5 by using caustic soda (50 wt.-% NaOH solution), before an
alcalase (Alcalase 2.4 FG
(Novozymes)) was added in liquid form in an amount of 0.5 wt.-% (by weight
broth). Stirring was
continued for 3 hours at 60 C. After that, the lysed cell mixture was
transferred into a forced circulation
evaporator (obtained from GEA, Germany) and heated to a temperature of 85 C.
The mixture was
concentrated in the forced circulation evaporator, until a total dry matter
content of about 30 wt.-% was
reached. The concentrated lysed cell mixture was transferred into a new
vessel, heated up to 90 C
under low shear agitation, while adjusting the pH to 10.5 by adding caustic
soda. Low shear agitation
was continued for about 30 hours, while keeping the temperature at 90 C and
the pH above 9.0 by
adding caustic soda.
After that the resulting demulsified mixture was neutralized by adding
sulfuric acid to adjust a pH of
7.5. Phase separation into a light phase, containing the oil, and a heavy
phase, containing water, cell-
debris, residual oil and salts, was carried out mechanically by using a disc
stack separator (Alfa Laval
Disc Stack Centrifuge, LAPX 404/Clara 20)
After separation of the crude oil, the remaining cell debris were resuspended
in the aqueous phase,
concentrated and dried by spraygranulation.
Due to the efficient demulsification, more than 90 wt.-% of the oil could be
separated from the biomass
without the addition of organic solvents or sodium chloride.
The remaining heavy phase was converted into a solid biomass by concentrating
via evaporation to a
total dry matter of 45 wt.-% at a temperature of about 90 C and subsequent
drying via spray
granulation in a fluidized bed spray granulator. The resulting biomass
exhibits a high bulk density of
more than 530 kg/m3, a high energy value of about 4000 kcal/kg and very good
handling properties, in
particular a flowability of 4. Comparable biomasses originating from
Schyzochitria as available on the
market exhibit all a much worse flowability of 6 and a much lower bulk density
of between 325 to 500
kg/m3.
.. In some aspects, embodiments of the present invention as described herein
include the following
items:
Item 1. A method of separating a polyunsaturated fatty acids (PUFAs)
containing lipid from the debris
of a biomass, comprising the following steps:
a) providing a suspension of a biomass comprising cells which contain a PUFAs
containing
b) lysing the cells of the biomass;
Date Regue/Date Received 2022-07-14
19
c) heating the suspension as obtained in step (b) to a temperature of 80 C to
100 C , while
adjusting the pH to a value of 9.0 to 11.5;
d) keeping the temperature and pH value in the ranges as depicted in (c) for
at least 10
hours, resulting in a demulsified composition;
e) harvesting of the PUFAs containing lipid from the demulsified composition
as obtained in
step d);
characterized in that after lysing of the cells, the suspension is
concentrated to a total dry
matter content of 30 to 60 wt. -% and no salts or less than 0.5 wt.-% of salts
are used for
isolating the lipid from the biomass.
Item 2. The method according to item 1, wherein the temperature in step c) is
of 85 C to 95 C.
Item 3. The method according to item 1, wherein the temperature in step c) is
of about 90 C.
Item 4. The method according to item 1, wherein the pH in step c) is 9.0 to
11Ø
Item 5. The method according to item 1, wherein the pH in step c) is 9.0 to
10.5.
Item 6. The method according to item 1, wherein step d) lasts 15 to 40 hours.
Item 7. The method according to item 1, wherein step d) lasts 20 to 36 hours.
Item 8. The method according to any one of items 1 to 7, wherein lysing of the
cells of the biomass is
carried out enzymatically, mechanically, chemically and/or physically.
Item 9. The method according to item 8, wherein lysing of the cells of the
biomass is carried out as
follows:
a) heating the suspension of the biomass to a temperature of between 50 C
and 70 C,
adding a cell wall-degrading enzyme to the suspension, and adjusting an
adequate pH value,
if necessary, at which the enzyme is properly working; and
b) keeping the temperature and pH in the ranges as depicted in (a) for at
least one hour.
Item 10. The method according to item 9, wherein the temperature in step i) is
between 55 C and
65 C.
Item 11. The method according to item 9, wherein step ii) lasts for at least
two hours.
Item 12. The method according to item 9, wherein step ii) lasts for two to
four hours.
Date Regue/Date Received 2022-07-14
20
Item 13. The method according to any one of items 9 to 12, wherein the cell-
wall degrading enzyme is
selected from proteases, cellulases, hemicellulases, chitinases, pectinases,
sucrases, maltases,
lactases, alpha-glucosidases, beta-glucosidases, amylases, lysozymes,
neuraminidases,
galactosidases, alpha-mannosidases, glucuronidases, hyaluronidases,
pullulanases,
glucocerebrosidases, galactosylceramidases, acetylgalactosaminidases,
fucosidases,
hexosaminidases, iduronidases, maltases-glucoamylases, beta-glucanases,
mannanases, and
combinations thereof.
Item 14. The method according to any of items Ito 13, wherein less than 1 wt.-
% of organic solvents
are used in the method.
Item 15. The method according to item 14, wherein less than 0.5 wt.-%, of
organic solvents are used
in the method.
Item 16. The method according to item 14, wherein less than 0.1 wt.-%, of
organic solvents are used
in the method.
Item 17. The method according to any one of items Ito 16, wherein harvesting
of the PUFAs
containing lipid comprises neutralization of the demulsified suspension and
subsequent separation of
the PUFAs containing lipid as a lipid oil containing light phase from a heavy
phase comprising water,
salts, residual oil and cell debris, wherein the heavy phase is a PUFAs
containing aqueous
suspension.
Item 18. The method according to item 17, wherein the separation is realized
by mechanical means.
Item 19. The method according to item 17 or 18, wherein the separation is
realized at a temperature
of 60-90 C and at a pH value of 6-9.
Item 20. The method according to any one of items 17 to 19, wherein the
separation is realized at a
temperature of 70-80 C.
Item 21. The method according to any one of items 17 to 20, wherein the
separation is realized at a
pH value of 7-8.5.
Item 22. The method according to any one of items 17 to 21, comprising as a
further step the
conversion of the heavy phase into a dried biomass by drying the heavy phase
to a total dry matter
content of more than 90 wt.- /0.
Item 23. The method according to any one of items 1 to 22, wherein the
suspension is provided as a
fermentation broth with a biomass density of at least 80 g/l.
Date Regue/Date Received 2022-07-14
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Item 24. The method according to item 23, wherein the biomass density is at
least 100 g/I.
Item 25. The method according to item 23, wherein the biomass density is at
least 120 g/I.
Item 26. The method according to item 23, wherein the biomass density is at
least 140 g/I.
Item 27. The method according to any one of items 1 to 26, wherein the cells
which contain the
PUFAs containing lipid are selected from algae, fungi, protists, bacteria,
microalgae, plant cells, and
mixtures thereof.
Item 28. The method according to item 27, wherein the microalgae are selected
from the phylus
Stramanopiles.
Item 29. The method according to item 27, wherein the microalgae are selected
from the family of
Thraustochytrids.
Item 30. The method according to item 27, wherein the microalgae are selected
from the genus
Schizochytrium.
Item 31. A PUFAs containing aqueous suspension obtained by the method
according to any one of
items 17 to 21, wherein the PUFAs containing aqueous suspension contains a
delipidated biomass,
characterized by a content of organic solvents of less than 0.1 wt.-%, and by
a content of chloride of
less than 0.5 wt.-%, wherein the biomass contains cells and/or cell debris of
the phylus Stramanopiles,
and wherein the aqueous suspension is characterized by a total dry matter
content of 30 to 60 wt.-%.
Item 32. A method of feeding animals, wherein the PUFAs containing aqueous
suspension according
to item 31 is provided to animals.
Item 33. A PUFAs containing lipid obtained by the method according to any one
of items 17 to 21,
wherein the PUFAs containing lipid has a content of crude fat of more than 90
wt.-%.
Date Regue/Date Received 2022-07-14
