[0001] The present invention relates to the treatment of microbial oils, in particular those
containing one or more polyunsaturated fatty acids (PUFAs). The treatment comprises
adding a solvent to the oil, and cooling the oil until a precipitate forms, and then
removing the precipitate. The resulting oil may be enriched in PUFAs.
[0002] Although microbial oils, for example containing PUFAs, are known, there is a need
to improve the quality of the oil, and in particular to increase the amount of PUFAs
in the oil. The oil can thus be made more concentrated, and so less oil may be required
in order to deliver a desired quantity of a PUFA. There is also a need to be able
to purify microbial and PUFA-containing oils sufficiently in order that they can be
incorporated into foodstuffs such as infant formula or other edible compositions,
such as pharmaceuticals. This purification process is desirably efficient and cost-effective.
[0003] WO-A-97/43362 (Gist-Brocades B.V.) describes the extraction of sterols from microbial
oils with a polar solvent. However, unlike the present invention, it does not include
the cooling of the oil (winterisation) or the formation of precipitate, which is then
removed.
[0004] A first aspect of the present invention thus relates to a process for treating a
microbial oil or an oil comprising an (e.g. Ω3 or Ω6) PUFA. This process may comprise
adding a precipitate inducer (or enhancer) to the oil. Cooling (of the oil and precipitate
inducer mixture) can then take place until a precipitate forms or at least part of
the oil solidifies. The precipitate (solid or solidified matter) may then be removed
(from the e.g. residual oil).
PUFAs and oils
[0005] Preferably the oil is a microbial oil or it comprises one or more PUFAs. It is usually
a liquid. Preferred PUFAs are C18, C20 or C22 (e.g. Ω6 or Ω3) PUFAs. Preferred Ω3
and Ω6 PUFAs include:
(Ω3) docosahexaenoic acid (DHA), suitably from algae or fungi, such as the dinoflagellate
Crypthecodinium or the fungus Thraustochytrium;
(Ω6) γ-linolenic acid (GLA);
(Ω3) α-linolenic acid (ALA);
(Ω6) dihomo-γ-linolenic acid (DGLA);
(Ω6) arachidonic acid (ARA); and
(Ω3) eicosapentaenoic acid (EPA).
[0006] The microbial oil may thus comprise an Ω3 or an Ω6 PUFA. The Ω3 PUFA (e.g. DHA)-containing
oil may be a marine, e.g. fish (such as tuna) oil. The Ω6 and/or Ω3 PUFA (e.g. ARA,
DHA or EPA)-containing oil can be a microbial or single cell oil.
[0007] An Ω6 and/or Ω3 PUFA-containing microbial oil (e.g. GLA, ARA and EPA) can be obtained
from fungi, such as
Mortierella,
Pythium or
Entomophthora. Ω3 PUFAs (e.g. EPA) can be produced from algae such as
Porphyridium or
Nitzschia.
[0008] Preferably the microbial (or Ω6 or Ω3 (e.g. ARA, DHA or EPA)) oil can be produced
by a single cell or a microorganism. This may be a bacteria, yeast, algae or fungi.
Preferred fungi are of the order
Mucorales. The fungus may be of the genus
Mortierella,
Phycomyces,
Blakeslea or
Aspergillus. Preferred fungi are of the species
Mortierella alpina,
Blakeslea trispora and
Aspergillus terreus.
[0009] Preferred yeasts are of the genus
Pichia or
Saccharomyces, for example
Pichia ciferrii. Bacteria can be of the genus
Propionibacterium. Preferred algae are dinoflagellate and/or belong to the genus
Crypthecodinium, for example are of the species
Crypthecodinium cohnii.
[0010] The Ω6 and/or Ω3 PUFA-containing oil may be an edible oil or a vegetable oil. These
include blackcurrant, borage and primrose oils, and often contain an Ω6 PUFA, e.g.
GLA. They also include olive, sunflower and soybean, soy flower oils, for example
cooking and/or salad oils.
Production of crude oils
[0011] The starting (e.g. crude) oil may be a microbial (e.g. single cell) oil, or it may
be a marine (e.g. fish) oil or vegetable oil (either crude or partially treated).
In particular, crude oils containing Ω3 PUFAs (DHA and/or EPA) can be marine oils.
If the PUFA oil is to contain GLA, then the crude oil may be a vegetable oil, for
example blackcurrant, borage, sunflower, soybean or primrose oil.
[0012] A number of documents describe the production of crude PUFA oils. Microbial oils
containing ARA are disclosed in WO-A-92/13086 (Martek), EPA in WO-A-91/14427 (Martek)
and DHA in WO-A-91/11918 (Martek). The present Applicant has already described various
methods for extracting PUFA oils from microbial sources, and these can be found in
WO-A-97/36996 and WO-A-97/37032 (both Gist-Brocades). Preparation of ARA, DHA and
EPA-containing oils is also described in WO-A-92/12711 (Martek).
In the oil, it is preferred that most of the PUFA is in the form of triglycerides.
Thus, preferably at least 50%, such as at least 60%, or optimally at least 70%, of
the PUFA is in triglyceride form. Of these triglycerides, preferably at least 40%,
such as at least 50%, and optimally at least 60% of the PUFA is present at the α-position
of the glycerol (present in the triglyceride backbone), also known as the 1 or 3 position.
It may be preferred that at least 20%, such as at least 30%, optimally at least 40%
of the PUFA is at the β(2) position.
[0013] Preferably the original microbial oil is a crude oil. It may have been extracted
from microbes or single cells, for example by using a solvent, such as supercritical
carbon dioxide, hexane or isopropanol. The oil may have been processed and/or treated
before the process of the present invention is performed.
Precipitate inducer
[0014] The inducer which is added to the microbial oil is preferably one that elevates or
increases the temperature at which the precipitate forms, for example the temperature
at which crystallisation starts. It may thus increase the crystallisation temperature
of the oil. The inducer may facilitate precipitation: without the inducer it has not
been found possible to form a precipitate (at least at temperatures down to about
-20°C, such as those found in freezers). With no inducer added, no precipitate formed
in a control experiment (see Comparative Example 5).
[0015] The inducer is preferably an (organic) liquid, although it may be polar or nonpolar.
Preferably the inducer will have a melting point of below the precipate, that is to
say a temperature bow that at which the precipitate forms. Thus the inducer preferably
has an m.p. below -70°C, such as below -80°C optimally below -90°C. The inducer is
suitably a clear liquid.
[0016] Suitably the inducer will be a solvent for (or miscible with) fats or oils, in particular
a solvent for (or miscible with) saturated triglycerides (that is to say, triglycerides
from saturated fatty acids) or a PUFA. Particularly preferred inducers are hexane
and/or acetone.
[0017] The inducer is preferably a liquid that is miscible with the oil, and hence a preferred
inducer is one that can dissolve or is miscible with a PUFA. Inducers that may thus
not be suitable are those that are immiscible with the oils, in particular alcohols
(e.g. methanol, isopropanol).
[0018] The precipitate inducer can therefore be regarded as a solvent for the oil to which
it is added. However, the inducer is preferably a non-solvent for at least one component
of the oil that is present in the precipitate, that is to say it does not dissolve
or it is immiscible with this component. This component which may be a single compound)
is one that is desired to be removed, and this is achieved by removing the precipitate
(or solidified matter) from the remaining oil. Although not wishing to be bound by
theory, it is thought that the precipitate inducer may bind to or surround the component
that is to be removed. The inducer may thus in same way face the component out of
solution (ie. out of the oil). However, whatever the mechanism, the precipitate inducer
clearly seems to play an important role in being able to facilitate the formation
of the precipitate. It appears to assist or induce precipitate formation, and such
a precipitate can contain at least one component that the inducer is not a solvent
for.
[0019] In the same way as one can describe compounds as hydrophilic and hydrophobic, one
can think of various components in the oil as being either inducer-liking (the inducer
is a solvent for that component) or inducer-hating (the inducer is a non-solvent for
that component). Once the precipitate performs, the precipitate may contain more inducer-hating
component(s) than the remaining oil. Thus, when the oil separates into two phases,
namely the remaining oil (usually on top) and the precipitate (usually on the bottom),
the oil will be enriched in an inducer-loving component (or depleted in an inducer-hating
component) while the precipitate will be enriched in an inducer-hating component (but
depleted in an inducer-loving component).
[0020] The ratio of oil:inducer is preferably from 1:1 to 1:10, such as from 1:2 to 1:9.
These ratios are particularly applicable if the solvent comprises acetone or hexane.
[0021] The inducer may be added to the oil when one or both of the oil and inducer are liquids,
such as at room temperature. However, addition can take place at any suitable temperature
of from 0-20°C, preferably from 0°C to 20°C, optimally 2°C to 30°C.
Cooling
[0022] The oil and inducer are mixed and then the mixture is allowed to cool. The mixture
is suitably homogeneous, i.e. a one-phase mixture. Cooling may take place naturally
or passively (for example by placing the mixture outside in a cool environment). However,
the oil can be actively cooled, for example using a heat exchanger.
[0023] Preferably however the oil is cooled by using a refrigerator or freezer. Cooling
may take place slowly. Preferably the oil and inducer mixture is placed in an environment
that is at a temperature below which a precipitate forms. This temperature is preferably
below 0°C or -5°C, such as below -10°C, suitably below - 20°C, and optimally at or
below -25°C. The time taken to cool the oil (and solvent mixture) may be from 1 to
30 hours, such as from 16 to 24 hours, optimally from 18 to 22 hours.
Nature of precipitate (or sediment) and its removal
[0024] The precipitate is usually a solid that forms due to cooling and for convenience
this term (including its use in "precipitate inducer") refers to the solidified matter
resulting from the lowering in temperature. Preferably the precipitate comprises crystals.
If so, then the inducer may increase the crystallisation temperature of the oil (or
one or more component(s) in the oil). The crystals may comprise only one component
or compound. The precipitate may thus comprise one or more impurities and/or unwanted
compounds, for example a saturated triglyceride. Solidification occurs at (or, to
put another way, the melting point of the solidified matter or precipitate is) preferably
from -1°C to -25°C, such as from -3°C to -20°C, preferably from -5°C to -17°C. Solidification
occurs at about - 15°C if the solvent is acetone and about -5°C if the solvent is
hexane.
[0025] Suitably the melting point of the precipitate is increased by the addition of the
inducer. With no inducer present, no precipitate was formed even when the oil was
cooled to -20°C (see Comparative Example 5).
[0026] Preferably the precipitate will not contain much PUFA, or at least only a small amount.
The amount of PUFA (e.g. ARA) in the precipitate is preferably less than 40%, such
as less than 35%, optimally less than 30% (by weight: 1% here is equivalent to 10g
PUFA/kg oil).
[0027] The precipitate is preferably denser than the oil. If this is so, then the precipitate
may fall or migrate to the bottom of the oil. It may thus be or form a sediment. In
this case the precipitate may be removed by centrifugation. This can take place in
a closed system, and so may minimise exposure of the oil to degrading substances,
such as oxygen in the atmosphere. Preferred centrifuges are laboratory or industrial
centrifuges. Centrifugation may take place at from 2000 rpm to 8000 rpm, such as from
3000 to 7000 rpm, optimally from 4000 to 6000 rpm. Put another way, centrifugation
may occur at from 2,000-8,000g, such as from 3,000-7,000g, optimally from 4,000-6,000g.
This may take place at from 1 to 4 minutes, such as from 12 to 3 minutes, preferably
for about 2 minutes.
[0028] Preferably the precipitate (or crystals) is removed by filtration. Here any crystals
formed may be sufficiently large enough to be removed by filtration. One can use a
plate and frame filter press or vacuum filtration. If using a filter press, the pressure
used is suitably from 0.2 to 0.5 bar. Removal may be by centrifugation or filtration
or both.
[0029] The cooling may thus result in a 2-phase system. A top layer may be liquid, such
as a residual oil. The bottom layer may be solid, and is thus the solidified matter
(or precipitate/sediment). Preferably the top layer is enriched in a PUFA while the
bottom layer is depleted in the PUFA. Thus, the residual oil may have a concentration
of the PUFA greater than the original oil and the solidified matter a reduced concentration
of the PUFA. This was unexpected as PUFAs often have a low m.p. and would be expected
to solidify first.
[0030] The residual oil may have a concentration of a PUFA (e.g. ARA) of at least 35%, such
as at least 37%, preferably at least 40%, optimally at least 42%.
[0031] Preferably, after the precipitate has been removed, the inducer is removed.
[0032] The inducer may be allowed to evaporate, for example at room temperature or above.
A suitable temperature is from 20 to 80 or 100°C, such as from 25 to 60°C, optimally
from 30-50°C. Removal of the inducer may take place with the aid of a vacuum.
[0033] As will be realised, since solidification usually takes place below 0°C, then warming
to room temperature may cause the solidified matter to melt and once again to become
liquid. Thus removal of the solidified matter preferably occurs while that matter
is still solid (or at a temperature below the melting point of that matter). Thus
removal preferably occurs at below 0°C, preferably below -5°C, optimally below -10°C.
[0034] Steps (b) - cooling - and (c) - removal of the solidified matter - can be repeated
at least twice, for example to enrich the oil further each time. Of course all the
steps (a) to (c) can be repeated, for example with the same or different inducer.
[0035] The (treated) oil resulting from the process of the invention, which forms the second
aspect of the invention, can be used for various purposes without further processing,
or can be additionally subjected to one or more purifying and/or refining steps. The
oil may thus be subjected to the purifying process described in European patent application
no. 00306606.5 filed on 2 August 2000.
[0036] The oil can be used as an additive or a supplement, for example in food compositions,
such as in infant formula. It may however also be used in cosmetic or pharmaceutical
compositions. The invention in a third aspect therefore relates to an edible composition,
such as a foodstuff, feed, pharmaceutical or cosmetic composition which comprises,
or to which has been added, the oil of the second aspect of the invention. Preferred
compositions are foods such as infant formula and nutritional supplements.
[0037] The oil of the invention can therefore have a relatively high PUFA content. This
may be at least 38%, preferably at least 40%, optimally at least 42% (by weight).
[0038] The oil is particularly suitable for nutritional purposes, and so can be used as
or in a nutritional supplement. The oil may be supplied as an oil, or it may be encapsulated,
for example, in a gelatin capsule. The oil can thus be incorporated into foods, feeds
or foodstuffs, suitable for animal or human consumption. Suitable examples are health
drinks and bread.
[0039] Preferred features and characteristics of one aspect of the invention are equally
applicable to another aspect
mutatis mutandis.
[0040] The invention will now be described, by way of example, with reference to the following
Examples. These are provided merely for means of illustration, and are not to be construed
being limiting on the invention.
EXAMPLE 1
[0041] Crude arachidonic acid (ARA) oil (100 ml) was obtained from
Mortierella alpina using hexane as the extracting solvent. The protocol for preparing such an oil is
described in Example 16 of WO-A-97/36996 (Gist-Brocades B.V.). This crude oil was
mixed with 400 ml of n-hexane. The resulting homogeneous mixture was placed in a freezer
held at a temperature of from -18°C to -25°C for 20 hours. A precipitate of crystals
formed at about -15°C. Hence a 2-phase system formed, with a top (liquid) layer (the
remaining oil) and a bottom layer (solid, precipitate). The precipitate was separated
using a laboratory centrifuge (type Sigma 4-10) at 5000 rpm for 2 minutes. The hexane
was removed from each of the remaining oil and the precipitate by evaporation overnight
at 30°C in a vacuum. The ARA content of the precipitate and residual oil was analyzed
by means of gas chromatography (GC) and the results are shown below in Table 1.
Table 1
Substance |
ARA (g/kg) |
Original oil |
345 |
Oil:precipitate |
317 |
Oil:residual oil |
406 |
EXAMPLE 2
[0042] Crude ARA oil (20 ml), from the same source as that in Example 1, was mixed with
80 ml of acetone. The resulting homogeneous mixture was placed in a freezer held at
a temperature of from -18°C to -25°C for 20 hours. A precipitate of crystals formed
at about -15°C to give a 2-phase system consisting of a top liquid layer and a bottom
solid layer. The precipitate was separated using a laboratory centrifuge (type Sigma
4-10) at 5000 rpm for 2 minutes. The hexane was removed separately from both the residual
oil and precipitate by evaporation overnight at 30°C in a vacuum. The ARA content
of both layers was analyzed by means of gas chromatography (GC) and the results are
shown below in Table 2.
Table 2
Substance |
ARA (g/kg) |
Original oil |
345 |
Oil:precipitate |
240 |
Oil:residual oil |
433 |
EXAMPLE 3
[0043] Different amounts of solvent (acetone) were added to the crude arachidonic oil used
in Example 1. The resulting homogeneous mixtures were placed in a freezer held at
a temperature of from -18°C to -25°C for 20 hours. A precipitate of crystals formed
at about -15°C to give a 2-phase system of a top layer (liquid) and a bottom layer
(solid, precipitate). The precipitate was separated using a laboratory centrifuge
(type Sigma 4-10) at 5000 rpm for 2 minutes. The hexane from both layers was removed
by evaporation overnight at 30°C in a vacuum and their ARA content was analyzed by
means of gas chromatography (GC) The results are shown below in Table 3.
Table 3
Ratio of Oil:acetone |
ARA (g/kg) |
|
Oil:top layer (residual oil) |
Oil:bottom layer (precipitate) |
Original oil |
358 |
1:1 |
364 |
351 |
1:2 |
399 |
326 |
1:3 |
394 |
296 |
1:4 |
421 |
261 |
EXAMPLE 4
[0044] Different amounts of solvent (acetone) were added to the crude arachidonic oil used
in Example 1. The resulting homogeneous mixtures were placed in a freezer held at
a temperature of from -18°C to -25°C for 20 hours. A precipitate of crystals formed
at about -15°C to give a 2-phase system consisting of a top layer (liquid, residual
oil) and a bottom layer (solid, precipitate). The precipitate was separated using
a laboratory centrifuge (type Sigma 4-10) at 5000 rpm for 2 minutes. The acetone was
removed from both layers separately by evaporation overnight at 30°C in a vacuum.
The ARA content of both layers was analyzed by means of gas chromatography (GC) and
the results are shown below in Table 4.
Table 4
Ratio of Oil:acetone |
ARA (g/kg) |
Yield |
|
Oil : top layer (residual oil) |
Oil: bottom layer (precipitate) |
(% of oil recovered from top layer) |
Original oil |
341 |
- |
1:3 |
374 |
265 |
59 |
1:4 |
382 |
224 |
72 |
1:5 |
383 |
204 |
76 |
1:6 |
371 |
191 |
77 |
1:7 |
372 |
185 |
77 |
1:8 |
368 |
183 |
77 |
1:9 |
368 |
178 |
78 |
Comparative Example 5
[0045] Arachidonic oil, from the same source as used in Example 1, was placed in a freezer
held at a temperature of from -18°C to-25°C for 20 hours (i.e. with no precipitate
inducer added). The oil was cooled to below -20°C. Surprisingly, even at -20°C, with
no inducer, no precipitate formed.
1. A process for treating a microbial oil or an oil comprising an Ω3 or Ω6 polyunsaturated
fatty acid (PUFA), the process comprising:
(a) adding a precipitate inducer to the oil;
(b) cooling until at least a part of the oil solidifies ; and
(c) removing the solidified matter (precipitate).
2. A process according to claim 1 wherein the oil comprises a C18, C20, C22, Ω6 or Ω3
PUFA and/or ARA, DHA, EPA or DGLA.
3. A process according to claim 1 or 2 wherein the oil is enriched in a PUFA and/or saturated
triglycerides are removed from the oil.
4. A process according to any preceding claim wherein the cooling results in the formation
of two layers, a top layer having a greater concentration of PUFA than the original
oil and the bottom layer (solidified matter) having a concentration of the PUFA less
than the original oil.
5. A process according to any preceding claim when the solidified matter is a precipitate
or sediment comprising crystals and/or the precipitate is removed either by centrifugation
or filtration.
6. A process according to any preceding claim wherein the inducer facilitates formation
of the precipitates by increasing the crystallisation temperature of the oil and/or
is a solvent for the PUFA.
7. A process according to any preceding claim wherein the inducer has a melting point
of at least -70°C and/or comprises acetone or hexane.
8. A process according to any preceding claim wherein the inducer is removed after the
precipitate has been removed.
9. A process according to any preceding claim wherein the ratio of oil:inducer is from
1:1 to 1:10 and/or removal of the solidified matter takes place at a temperature below
0°C.
10. A process according to any preceding claim wherein steps (b) and (c) are performed
at least twice and/or the solidifying starts at -5°C.