U.S. patent application number 12/242997 was filed with the patent office on 2009-02-26 for preparation of microbial oil.
This patent application is currently assigned to DSM IP Assets B.V.. Invention is credited to Petrus Joseph Maria Brocken, Hugo STREEKSTRA.
Application Number | 20090053342 12/242997 |
Document ID | / |
Family ID | 30773251 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090053342 |
Kind Code |
A1 |
STREEKSTRA; Hugo ; et
al. |
February 26, 2009 |
PREPARATION OF MICROBIAL OIL
Abstract
The present invention provides a process for the production of a
microbial oil comprising culturing a micro-organism in a two stage
fermentation process where, in a last stage that precedes the end
of fermentation, the carbon source is: consumed by the
micro-organisms at a rate greater than it is added to the medium;
added at a rate #0.30 M carbon/kg medium; or is rate limiting on
the growth of the micro-organism. The micro-organisms thus have the
carbon source restricted so that they preferentially metabolise
fats or lipids other than arachidonic acid (ARA), so increasing the
proportion of ARA in the cells. A microbial oil is then recovered
from the micro-organism, using hexane as a solvent, that has at
least 50% ARA and at least 90% triglycerides.
Inventors: |
STREEKSTRA; Hugo;
(Amsterdam, NL) ; Brocken; Petrus Joseph Maria;
(De Lier, NL) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
DSM IP Assets B.V.
HEERLEN
NL
|
Family ID: |
30773251 |
Appl. No.: |
12/242997 |
Filed: |
October 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10518949 |
Dec 17, 2004 |
7470527 |
|
|
PCT/EP2003/006552 |
Jun 20, 2003 |
|
|
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12242997 |
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Current U.S.
Class: |
424/780 ;
426/601; 435/134; 554/8 |
Current CPC
Class: |
A61K 8/9728 20170801;
A61P 25/00 20180101; A61K 8/67 20130101; C12P 7/6481 20130101; A23D
9/02 20130101; A61P 3/02 20180101; A23D 9/00 20130101; A61Q 19/00
20130101; A23L 33/115 20160801; C12N 1/005 20130101; A61K 8/925
20130101; A23L 33/12 20160801; A61K 2800/85 20130101; A23K 20/158
20160501; C11B 1/10 20130101; C12P 7/6427 20130101; C12P 7/6472
20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/780 ;
435/134; 554/8; 426/601 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12P 7/64 20060101 C12P007/64; A61P 43/00 20060101
A61P043/00; A23D 7/00 20060101 A23D007/00; C11B 1/00 20060101
C11B001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 19, 2002 |
EP |
02254262.5 |
Dec 18, 2002 |
EP |
02258713.3 |
Feb 26, 2003 |
EP |
03251169.3 |
Claims
1. A process for the production of a polyunsaturated fatty acid
(PUFA), the process comprising culturing a micro-organism in a
culture medium inside a fermentation vessel, whereby at a stage
which precedes the end of fermentation; a) the carbon source is
consumed by the micro-organisms at a rate greater than it is added
to the medium; b) the carbon source is added at a rate of # 0.30M
carbon/kg medium per hour; c) the carbon source is rate limiting on
the growth of the micro-organisms, or is restricted such that the
micro-organisms metabolise fat(s) and/or lipid(s); d) the rate of
addition of the carbon source is reduced or is below the rate of
consumption of the carbon source by the micro-organisms; or e) the
carbon source has been all used, or has a concentration in the
medium of about zero, at or before the end of fermentation; f) the
carbon source addition is stopped but fermentation is allowed to
continue; and/or g) the micro-organisms are subjected to conditions
whereby they metabolise, or consume, one or more fat(s) or
lipids(s) in preference to arachidonic acid (ARA).
2. A process according to claim 1, wherein: h) the concentration of
the carbon source in that stage is on average # 10 g/kg and/or #
0.17 M carbon/kg medium; i) the carbon source is glucose; and/or j)
the PUFA is present in a microbial oil.
3. A process according to claim 2, wherein: k) the second stage
starts at from 15 to 2 hours before the end of the fermentation or
less than 10 days after the beginning of the fermentation.
4. A process according to claim 3, wherein: l) the (entire)
fermentation is carried out at a temperature .E-backward.22.degree.
C. or and/or <30.degree. C.; m) it is conducted in the absence
of an additive oil; and/or n) the fermentation lasts no longer than
9 days.
5. A process according to claim 4, wherein: n) the PUFA comprises
arachidonic acid (ARA); and/or o) the vessel has a capacity of at
least 10 litres.
6. A process according to claim 1 wherein the micro-organism is
Mortierella, optionally Mortierella alpina, and/or is
non-genetically modified.
7. A microbial oil which comprises at least 50% arachidonic acid
(ARA) and: a) has at least a 90% triglyceride content; b) has a
peroxide value (POV) of no more than 2.5; c) has an anisidine value
(AnV) of no more than 1.0; d) is hexane extracted; and/or e) has a
phospholipid content below 5%.
8. An oil according to claim 7 which comprises: f) less than 5% of
C.sub.20 and/or C.sub.24 polyunsaturated fatty acids (PUFAs);
and/or g) less than 5% of C.sub.22+ PUFAs.
9. An oil according to claim 8 wherein: h) the free fatty acid
content is .ltoreq.0.4%; i) the triglyceride content is at least
95% or 98%.
10. A composition comprising a microbial oil obtainable by a
process according to claim 1.
11. A composition according to claim 12 which is a foodstuff, food,
feed, or feed supplement, pharmaceutical, veterinary or cosmetic
composition.
12. A composition comprising a microbial oil of claim 7.
13. An infant formula composition comprising a composition
according to claim 12.
Description
RELATED APPLICATIONS
[0001] This is a divisional of application Ser. No. 10/518,949,
filed Dec. 17, 2004 (allowed), which is a U.S. national phase of
PCT/EP2003/006552, filed Jun. 20, 2003, which claims benefit of
European Applications 03251169.3, filed Feb. 26, 2003, 02258713.3,
filed Dec. 18, 2002 and 02254262.5, filed Jun. 19, 2002, the entire
contents of each of which is hereby incorporated by reference in
this application.
FIELD OF THE INVENTION
[0002] The present invention relates to a process for the
production of a PUFA, optionally in a microbial oil, comprising
culturing a micro-organism in a two-stage process and subsequently
recovering the microbial oil from the micro-organisms. The
invention also relates to a novel (e.g. microbial) oil resulting
from such a process. In the oil 50% or more of the lipids (or
PUFAs, such as in the oil) are arachidonic acid (ARA). The oil may
have a low peroxide value (POV), of below 2.5 or 2.0 and/or a low
anisidine value (AnV), below 1.0. The present invention also
relates to foodstuffs and food supplements comprising, or generated
using, the microbial oil of the invention.
INTRODUCTION
[0003] Polyunsaturated fatty acids, or PUFAs, are found naturally.
A wide variety of different PUFAs are produced by different single
cell organisms (algae, fungi, etc). One particularly important PUFA
is arachidonic acid (ARA), one of a number of Long Chain
Poly-Unsaturated Fatty Acids (LC-PUFAs). Chemically, archidonic
acid is cis-5,8,11,14 eicosatetraenoic acid (20:4) and belongs to
the (n-6) family of LC-PUFAs.
[0004] Arachidonic acid is a major precursor of a wide variety of
biologically active compounds, known collectively as eicosanoids, a
group comprising prostaglandins, thromboxanes and leukotrienes.
Arachidonic acid is also one of the components of the lipid
fraction of human breast milk and is thought to be essential for
optimal neurological development in infants. Arachidonic acid has a
wide variety of different applications including use in infant
formula, foodstuffs and animal feeds.
[0005] Arachidonic acid can be produced using micro-organisms and
in particular using the filamentous fungi Mortierella. However, the
percentage of arachidonic acid in the microbial oil produced is
usually too low. A number of attempts have been made to try and
increase the yield of arachidonic acid from Mortierella, but with
varying degrees of success. Many of the attempts to increase
arachidonic acid levels have involved steps that cannot be readily
used in an industrial setting.
[0006] For example, Eroshin et al, Process Biochemistry: (35) 2000,
pp 1171-1175 leaves the culture to sit for a period of about a week
after the end of fermentation. The amounts of ARA quoted are based
on the biomass (and not on oil extracted from it) as this document
does not describe the extraction of any oil. Totani et al,
Industrial Applications of single cell oils, American Oil Chemists'
Society Campaign, 1992, Chapter 4, pp 52-60 and Lipids, vol. 22 No.
12 (1987) pages 1060-1062 advocates the use of an unusually low
fermentation temperature which means the fermentation is
considerably slowed. The ARA content here is based on extraction
with a chloroform/methanol solvent mixture. Another document in the
ARA production field is WO 96/21037.
[0007] EP-A-1035211 (Suntory) describes a process for producing ARA
and DHGLA lipids from M. alpina. However, the calculation of ARA
content is based either on the biomass (rather than an oil
extracted from it), or results from an analytical method where the
polyunsaturated fatty acids are esterified first, and then
extracted using a solvent (rather than being extracted first, to
produce an oil, and the ARA content then being determined on the
basis of this oil).
[0008] One report of a higher yield of ARA alleges a concentration
in harvested mycelia of nearly 70% using the strain 1S-4 of M.
alpina (Shimizu S., Oils-Fats-Lipids 1995, Proc. World Congr. Int.
Soc. Fat Res., 21.sup.st (1996), Meeting Date 1995, Volume 1, pages
103-109 and Biochemical and Biophysical Research communications,
Vol. 150(1), 1988, pages 335-441). However this percentage is based
on the cells, and is not the same as the ARA percentage in a
microbial oil. In fact the oil made only gave an ARA content of
39.0% (Table 27.2, page 105). (Note that the art uses a variety of
different ways to measure ARA content, which may not necessarily be
the same unit or based on the same analytical protocol as the
figures quoted later in this specification). Furthermore, this was
only obtained when the M. alpina cells were allowed to stand at
room temperature for a further 6 days after fermentation, which is
clearly not a viable option for industrial production
processes.
[0009] There is, therefore, a need to identify ways to increase the
proportion (and so yield) of arachidonic acid in the microbial oils
and in particular in a way that can be employed on an industrial
scale.
SUMMARY OF THE INVENTION
[0010] The present invention provides novel processes to produce a
(microbial) oil with an increased proportion of arachidonic acid.
This means arachidonic acid may be produced at a reduced cost and
increased rate. In addition, as the present invention does not rely
on the genetic modification of micro-organisms involved in order to
enhance arachidonic acid production, the invention can help to meet
the increasing demand for natural, non-genetically modified food
ingredients. In addition the oil has low oxidation potential and so
is suitable for incorporation into human foods for which toxiciy is
particularly important, such as infant formula.
[0011] Accordingly, a first aspect of the invention relates to a
process for the production of a microbial oil or a polyunsaturated
fatty acid (PUFA). The process comprises fermenting (or culturing)
a micro-organism inside a fermentation vessel, suitably in a
culture medium, whereby before (or at a stage which precedes) the
end of fermentation; [0012] a) the carbon source is consumed by the
micro-organisms at a rate greater than it is added to the medium;
[0013] b) the carbon source is added at a rate of # 0.30M carbon/kg
medium per hour; [0014] c) the carbon source is rate limiting on
the growth of the micro-organisms, or is restricted such that the
micro-organisms metabolise (its own) fat(s) and/or lipid(s); [0015]
d) the rate of addition of the carbon source is reduced to, or is
below the, rate of consumption of the carbon source by the
micro-organisms; [0016] e) the carbon source has been all used, or
has a concentration in the medium of about zero, at or before the
end of fermentation; [0017] f) the carbon source addition is
stopped but fermentation is allowed to continue; and/or [0018] g)
the micro-organisms are subjected to conditions such that they
metabolise fat(s) (e.g. inside the cell, such as a PUFA) other than
ARA in preference to ARA.
[0019] A second aspect of the present invention relates to a
microbial oil which comprises at least 50% (or at least 50.5, 51 or
52%) arachidonic acid (ARA). It may have up to 55, 57 or 60% ARA.
This oil may have:
[0020] a) a triglyceride content of at least 90%;
[0021] b) a POV of less than 2.5;
[0022] c) an AnV of less than 1.0; and/or
[0023] d) a phospholipid content below 5%.
[0024] The oil can be preparable by the process of the first
aspect. It may be hexane extracted.
[0025] It is thought that once the concentration of carbon source
is reduced or restricted, the cells start metabolising the fats or
lipids inside the cell. However, the cells consume fats other than
ARA first. In this way the proportion of ARA in the fats or lipids
in the cells increases. Hence the process of the first aspect can,
in this way, lead to a higher ARA content oil of the second
aspect.
DETAILED DESCRIPTION OF THE INVENTION
Micro-organisms
[0026] The micro-organism employed may be a bacteria, yeast, algae
or fungus. Preferably a fungus is used and in particular a
filamentous fungus is used. Preferred fungi are of the order
Mucorales. The fungus may be of the genus Mortierella, Phycomyces,
Entomophthora, Pythium, Thraustochytrium, Blakeslea, Rhizomucor or
Aspergillus. Preferred fungi are of the species Mortierella alpina.
Preferred yeasts are of the genus Pichia or Saccharomyces, for
example Pichia ciferrii. Bacteria can be of the genus
Propionibacterium. Suitable algae are dinoflagellate and/or belong
to the genus Crypthecodinium, Porphyridium or Nitschia, for example
are of the species Crypthecodinium cohnii.
[0027] The micro-organism strains used in the present invention may
be a naturally occurring or commonly used industrial strain. The
strain may not have been genetically altered for example, it may
not be transformed with a vector nor may it contain heterologous
gene(s). Given the current preference in some quarters for
foodstuffs which do not contain genetically engineered ingredients,
the micro-organism employed may be a strain which has not been so
modified.
Polyunsaturated Fatty Acids (PUFAs)
[0028] The PUFA can either be a single PUFA or two or more
different PUFAs.
[0029] The or each PUFA can be of the n-3 or n-6 family. Preferably
it is a C18, C20 or C22 PUFA. It may be a PUFA with at least 18
carbon atoms and/or 3 or 4 double bonds. The PUFA(s) can be
isolated in the form of a free fatty acid, a salt, as a fatty acid
ester (e.g. methyl or ethyl ester), as a phospholipid and/or in the
form of a mono-, di- or triglyceride.
[0030] Suitable (n-3 and n-6) PUFAs include:
[0031] docosahexaenoic acid (DHA, 22:6 .OMEGA.3), suitably from
algae or fungi, such as the (dinoflagellate) Crypthecodinium or the
(fungus) Thraustochytrium;
[0032] .gamma.-linolenic acid (GLA, 18:3 .OMEGA.6);
[0033] .alpha.-linolenic acid (ALA, 18:3 .OMEGA.3);
[0034] conjugated linoleic acid (octadecadienoic acid, CLA);
[0035] dihomo-.gamma.-linolenic acid (DGLA, 20:3 .OMEGA.6);
[0036] arachidonic acid (ARA, 20:4 .OMEGA.6); and
[0037] eicosapentaenoic acid (EPA, 20:5 .OMEGA.3).
[0038] Preferred PUFAs include arachidonic acid (ARA),
docosohexaenoic acid (DHA), eicosapentaenoic acid (EPA) and/or
.gamma.-linoleic acid (GLA). In particular, ARA is preferred.
Fermentation
[0039] The fermentation/culturing will typically be carried out in
a suitable fermenter (or fermentation vessel) containing a (liquid,
usually aqueous) culture medium. A main fermenter vessel will
normally be aseptically inoculated from a small feed fermenter.
Typically a submerged and/or aerobic fermentation process is
employed. This may take place in a deep tank fermenter. The
fermenter may be equipped with devices to monitor and/or change pH
and temperature. The vessel may additionally be adapted to perform,
or allow to be conducted, aeration and/or mixing of the cells and
liquid, such as agitation of the solution. This may be stirring,
for example achieved using mechanical means.
[0040] Suitably the volume of the fermenter is at least 10, 20, 40
or even 60 m.sup.3. Volumes as high as 100 or even 150 m.sup.3 can
be used.
[0041] The fermentation will typically last for 10 days or less,
preferably 9 or less days, more preferably 8 or less days. It may
be at least 4, 5, 6 or 7 days.
[0042] Optionally the fermentation may be for 150 to 200 hours,
such as 160 to 190 hours, eg. from 170 to 180 hours. The end of
fermentation is usually the point at which agitation and/or
aeration is stopped. This can be when the fermenter vessel, and/or
ancillary equipment, is (effectively) switched off. The
micro-organisms may then be removed from the fermenter.
[0043] The fermentation may be at a temperature of from 20 to
40.degree. C.
Carbon and Nitrogen Sources
[0044] Any suitable medium may be used in the fermentation, for
example a medium appropriate to the micro-organism being used. The
carbon source can comprise (complex sources such as) maltodextrin,
oat flour, oat meal, molasses, vegetable (e.g. soy bean) oil, malt
extract, starch, ethanol or soy bean oil. Preferred (non-complex)
carbon sources include carbohydrates or sugars, such as fructose,
maltose, sucrose, xylose, mannitol, glucose or lactose or glycerine
(e.g. from a vegetable source), citrate, acetate, glycerol, ethanol
or (e.g. sodium) ascorbate. In a preferred embodiment of the
invention the carbon source is or comprises glucose, and in
particular is glucose syrup.
[0045] Suitable nitrogen sources include yeast extract, urea and
peptone. The medium can exclude agar.
[0046] Preferred nitrogen and/or carbon sources are water soluble
or water miscible.
[0047] Individual components of the medium (such as the nitrogen
and/or carbon sources) may either (all) be present at the start of
fermentation, added continuously during fermentation or added in
stepwise or batches. In particular, the amount of carbon source
present in the medium will typically be controlled as described
below, preferably by controlling the rate of addition of the carbon
source.
[0048] The nitrogen and/or carbon sources can be supplied (or
added) separately, or supplied simultaneously, or supplied as a
combined preparation. They may thus present in the same composition
(if thought necessary) which is preferably a liquid. The carbon
and/or nitrogen sources can be added (to the fermenter vessel)
either before the fungal cells are added (to the vessel), in other
words prior to inoculation, or during fermentation alternatively
they may be added both before fermentation and during.
Culture Medium
[0049] The culture medium is preferably an aqueous liquid. This may
additionally contain other substances to assist in the
fermentation, for example a chelating agent (e.g. citric acid), an
anti-foaming agent (e.g. soy bean oil), a vitamin (e.g. thiamine
and/or riboflavin), any necessary catalytic metals (for example,
alkali earth metals such as magnesium or calcium, or zinc or iron
and/or other metals such as cobalt and copper), phosphorus (e.g.
phosphate) and/or sulphur (e.g. sulphate). The medium may, if
necessary, contain an additive oil such as olive or soybean oil,
however preferably the medium does not contain such an oil.
[0050] The (optimum) growth (or fermentation) temperature may vary
depending on the micro-organism used. However, it is preferably
from 20 to 40.degree. C. and more preferably from 22 to 30 or
32.degree. C. In particular the temperature the fermentation is
carried out at will be .gtoreq.22.degree. C. or .ltoreq.25.degree.
C., eg. 22 to 30.degree. C., such as from 23-28.degree. C. The pH
of the aqueous liquid during fermentation may be from 4 to 10, such
as from 5 to 8, optimally from 6 to 7.
[0051] The medium will typically be stirred or agitated during
fermentation to help facilitate aeration. The aqueous liquid and
the cells are suitably either mixed or agitated. This may be
achieved if aeration is provided, such as by bubbling a gas, e.g.
air, into the aqueous liquid. This may serve the additional purpose
of providing oxygen to the fungal cells: hence the fermentation is
preferably an aerobic one. Other means of agitation or mixing
include stirring, for example using an impeller. This may be of a
hydrofoil axial flow design or may be designed so that the aqueous
medium is forced radially outwards from the impeller (such as a
turbine). Even if there is no stirring it is preferred that the
microbial cells are provided with oxygen during fermentation, and
so aeration (e.g. by bubbling air, oxygen or other
oxygen-containing gas) is advantageous here. Aeration may be at
from 0.1 to 2.0, such as from 0.5 to 1.0 vvm.
[0052] Preferably the volume of the fermenter is at least 2 or 5
litres, preferably at least 10 litres. However, for fermenters used
in industry, or on an industrial scale, the vessel volume is
preferably at least 50, 100, 500 or 1,000 litres.
Last (or Second) Stage of Fermentation
[0053] The fermentation process may be split into at least two
stages. A second or last stage, which may immediately precede the
end of fermentation, can be characterised by a decrease in the
amount of the carbon source available to the micro-organism or any
of the features (a) to (g) as given in the first aspect. Typically,
this stage can begin from 15 to 2 hours before the end of
fermentation, preferably less than or at 10 hours from the end of
fermentation, more preferably from 3 to 5 hours from the end of
fermentation. Preferably, this stage will typically begin less than
10 days after the beginning of fermentation, more preferably it
will begin less than 9 days after, even more preferably less than 8
days after.
[0054] During a first or earlier stage of fermentation the carbon
source may be in excess. Thus the amount of carbon source available
may not be limiting to on the growth of the micro-organisms. The
rate of addition of the carbon source may exceed the rate of its
consumption by the micro-organisms. In the second or last stage of
fermentation the amount of the carbon source being added can be
decreased or stopped altogether. This means that the amount of
carbon source available to the micro-organism will decrease during
the second or last stage of fermentation. Typically in a second,
final or last stage, or towards the end of fermentation, the carbon
source can be: [0055] consumed by the micro-organisms at a rate
greater than it is added to the medium (for example the rate of
addition is less than the rate of consumption); [0056] added at a
rate # 0.30 M carbon/kg medium per hour, such as #0.25 or #0.20,
but at least 0.01, 0.02 or 0.05 M carbon/kg medium/hr (the units
here referring to the moles, or molar amount, of carbon in the
carbon source, rather than the weight or moles of the carbon source
itself); [0057] rate limiting on the growth and/or (PUFA)
production of the micro-organisms.
[0058] Typically, the concentration of the carbon source during the
second stage is # 10 g carbon source/kg of medium, preferably from
0.01 or 0.1 to 8 or 10 g/kg, more preferably from 0.5 to 5 g/kg and
even more preferably from 1 or 2 to 4 or 5 g/kg. This means that,
on average during the last stage of fermentation, there will be #
0.30M carbon per kg of medium, preferably from 0.003 M to 0.3M
carbon per kg. Advantageously this is from 0.015M to 0.17M carbon
per kg and even more preferably from 0.03M to 0.17M carbon per
kg.
[0059] When the carbon source comprises glucose, typically the
concentration of glucose (in the last stage) will be on average #10
g/kg of medium, preferably from 0.01 or 0.1 to 8 or 10 g/kg.
Advantageously this is from 0.5 to 5 g/kg and even more preferably
from 1 or 2 to 4 or 5 g/kg medium. In this sense medium includes
the cells and the aqueous culture medium, that is to say it is the
"broth" (cells and surrounding liquid).
[0060] The rate of addition of the carbon source in the last stage
is preferably no more than 0.03M carbon per kg, preferably no more
0.025 or 0.02 M carbon per/kg (medium). Preferably the rate of
addition is about 0.015M carbon per/kg. If the carbon source is
glucose, then preferably the rate of addition of glucose is less
than 1.0, for example less than 0.8, for example less than 0.5 g
glucose per/kg medium per hour.
[0061] Preferably the rate of addition of the carbon source in the
last stage is approximately half that of the rate of consumption of
the carbon source by the micro-organisms. However, the ratio of
rate of addition: rate of consumption may vary from 1:1-3, such as
from 1:1.5 to 2.5, optimally from 1:1.8 to 2.2. Alternatively, the
rate of addition may be from 30-70%, such as from 40 to 60%,
optimally from 45 to 55%, of the rate of consumption.
[0062] The appropriate concentration of the carbon source during
the second stage of the fermentation can be achieved by carefully
controlling the rate of addition of the carbon source. Typically,
this will be decreased as appropriate during, or to precipitate the
onset of, the last stage. Periodic sampling and analysis of the
culture can be used to determine the concentration of the carbon
source and to make adjustments as necessary to the rate of addition
of the carbon source. This may be done automatically using a
computer system.
Pasteurisation Process
[0063] Pasteurisation will usually take place after fermentation
has finished. In a preferred embodiment, pasteurisation will finish
the fermentation, because the heat during pasteurisation will kill
the cells. Pasteurisation may therefore be performed on the
fermentation broth (or the cells in the liquid (aqueous) medium),
although it can be performed on the microbial biomass obtained from
the broth. In the former case, pasteurisation can take place while
the microbial cells are still inside the fermenter. Pasteurisation
preferably takes place before any further processing of the
microbial cells, for example granulation (e.g. by extrusion)
crumbling, or kneading.
[0064] Preferably the pasteurisation protocol is sufficient to
inhibit or inactivate one or more enzymes that can adversely affect
or degrade a PUFA or microbial oil, for example a lipase.
[0065] Once fermentation has been finished, the fermentation broth
may be filtered, or otherwise treated to remove water or aqueous
liquid. After water removal, one may obtain a biomass "cake". If
pasteurisation has not taken place, then the dewatered cells (or
biomass cake) can be subjected to pasteurisation.
Oil Extraction
[0066] If desirable, and for example after fermentation is
finished, the micro-organisms may be killed or pasteurised. This
may be to inactivate any undesirable enzymes, for example enzymes
that might degrade the oil or reduce the yield of the PUFAs.
[0067] After culturing or fermentation is complete or has ended,
the fermentation broth (cells and aqueous liquid) may then be
removed from the fermenter, and if necessary liquid (usually water)
removed therefrom. Any suitable solid liquid separation technique
can be used. This (dewatering) may be by centrifugation and/or
filtration. The cells may be washed, for example using an aqueous
solution (such as water) for example to remove any extracellular
water-soluble or water-dispersible compounds. An oil can then be
recovered from the microbes, for example using a solvent so that
the oil may be solvent-extracted, preferably hexane-extracted.
[0068] The oil may have no (or be substantially free from) GLA
and/or DGLA.
PUFA Extraction Process
[0069] The PUFA (or microbial oil, usually containing the PUFA) may
be extracted from (e.g. dried) granules (e.g. extrudates)
containing the cells. The extraction can be performed using a
solvent. Preferably a non-polar solvent is used, for example a
C.sub.1-8, e.g. C.sub.2-6, alkane, for example hexane. One may use
carbon dioxide (in a liquid form, for example in a super critical
state).
[0070] The cells may thus be subjected to extraction, such as with
an organic solvent, preferably under nitrogen flow. Other usable
organic solvents include ether, methanol, ethanol, chloroform,
dichloromethane and/or petroleum ether. Extraction with methanol
and petroleum ether and/or extraction with a one-layer solvent
system consisting of chloroform, methanol, and water can also be
used. Evaporation of the organic solvent(s) from the extract under
reduced pressure can give a microbial oil containing arachidonic
acid at a high concentration.
[0071] Preferably, the solvent is allowed to percolate over the
dried granules. Suitable micro-organism granulation and extrusion
techniques and subsequent extraction of a microbial PUFA containing
oil, are described in WO-A-97/37032.
[0072] The solvent allows one to obtain a crude PUFA containing
oil. This oil can be used in that state, without further
processing, or it can be subjected to one or more refining steps.
Suitable refining protocols are described in International patent
application no. PCT/EP01/08902 (the contents of this document and
all others described herein are hereby incorporated by reference).
For example, the oil can be subjected to acid treatment or
degumming, alkali treatment or free fatty acid removal, bleaching
or pigment removal, filtration, winterisation (or cooling, for
example to remove saturated triglycerides), deodorising (or removal
of free fatty acids) and/or polishing (or removal of oil-insoluble
substances).
[0073] The resulting oil is particularly suitable for nutritional
purposes, and can be added to (human) foods or (animal) feedstuffs.
Examples include milk, infant formula, health drinks, bread and
animal feed.
Purification/Refinement
[0074] The microbial oil may be refined and purified. This may
involve removing one or more of the following components: a
phospholipid, trace metal, pigment, carbohydrate, protein, free
fatty acid (FFA), oil insoluble substance, water insoluble
substance, soap or saponified substance, oxidation product,
sulphur, mono- or diglyceride, pigment decomposition product,
solvent and/or sterol. The purifying may reduce or remove
"off-flavours" and/or improve the stability of the oil.
[0075] To effect this the process (e.g. purifying) may comprise
degumming (or acid treatment), neutralization (or alkali
treatment), water washing, bleaching, filtering, deodorising,
polishing and/or cooling (or winterization). Preferably the
purifying comprises acid treatment and/or alkali treatment
(degumming and neutralisation). Alternatively purifying methods may
comprise bleaching and/or deodorization. Preferably however the
purifying will involve bleaching and/or deodorization, and
optimally in addition acid and/or alkali treatment.
Oils
[0076] The second aspect of the present invention provides a
microbial oil which comprises 35 or 40% of at least one PUFA, such
as ARA. The oil can have at least 50, 55 or 60% or more of this
PUFA, such as ARA. It can have triglyceride content of at least
90%. Preferably the microbial oil comprises from 50, 55 or 60 to
90% arachidonic acid, more preferably from 60 to 80% and even more
preferably from 60 to 70% arachidonic acid.
[0077] Preferably the microbial oil has a triglyceride content of
from 90 to 100%, such as at least 90 or 96%, preferably at least
98%, more preferably at least 99% and optimally above 99.5%.
Typically, the microbial oil will have an eicosapentaenoic acid
(EPA) content of below 5%, preferably below 1% and more preferably
below 0.5%. The oil may have less than 5%, less than 2%, less than
1% of each of C.sub.20, C.sub.20:3, C.sub.22:0 and/or C.sub.24:0
polyunsaturated fatty acid (PUFAs). The free fatty acid (FFA)
content may be #0.4, 0.2 or 0.1%.
[0078] Of the triglycerides, preferably at least 40%, such as least
50%, and more preferably at least 60% of the PUFAs present are at
the V-position of the glycerol (present in the triglyceride
backbone) also known at the 1 or 3 position. It is preferred that
at least 20%, such as least 30%, more preferably at least 40% of
the PUFA(s) is at the .E-backward.(2) position.
[0079] The phospholipid content of the oil is suitably at a maximum
of 5%, 3% or 2% and/or may be at a minimum of from 0.1, 0.5 or
1.0%.
[0080] Typically the microbial oil will be one obtainable by the
process of the first aspect invention. Preferably the oil will have
been isolated from a fungus, more preferably the oil is isolated
from Mortierella and in particular from M. alpina. The oil is
suitably hexane extracted.
ARA Content
[0081] Purely for the sake of clarity, the calculation of the
percentage ARA content will be explained, especially as the
literature can, on occasions, calculate the ARA content on a
different basis. The percentage of ARA is based on the oil (that
has been extracted from the biomass), and not the biomass itself.
It is on a weight by weight basis. It is based on an oil extracted
by hexane, and is therefore based on hexane extractable lipids
(HEL). It is based on the total amount of oil, and not on the total
amount of fatty acids (which can sometimes give a misleading higher
figure). The ARA content is determined by the well-known FAME
analytical protocol (using the fatty acid methyl esters), detailed
in AOCS Ce1 b89. Different solvents will extract different lipids.
Note that in the present case, the oil is first extracted with
hexane, and then the ARA content of the oil determined by FAME
analysis. This will give a different result from first esterifying
the arachidonic acid (e.g. while still in the cells) and then
extracting the resulting methyl esters for further analysis.
Peroxide Value (POV)
[0082] Preferably the POV of the microbial oil is no more than 3.0,
2.5 or 2.0. However, much lower POV values can be obtained using
the process of invention, and these values may be less than 1.5 or
less than 1.0. Values less than 0.8 and even less than 0.4 can be
obtained.
Anisidine Value (AnV)
[0083] Preferably the anisidine value of the microbial oil is no
more than 1.0, for example no more than 0.6, 0.3 or even no more
than 0.1.
Uses and Products
[0084] A third aspect of the invention relates to a composition
comprising the oil of the second aspect, and where appropriate one
or more (additional) substances. The composition may be a foodstuff
and/or a food supplement for animals or humans. In embodiments of
the invention which are for human consumption the oils may be
rendered suitable for human consumption, typically by refining or
purification of the oil obtained from the microbes.
[0085] The composition may be infant formula or (human) foodstuffs.
Here the composition of the formula may be adjusted so it has a
similar amount of lipids or PUFAs to normal breast milk. This may
involve blending the microbial oil of the invention with other oils
in order to attain the appropriate composition.
[0086] The composition may be an animal or marine feed composition
or supplement. Such feeds and supplements may be given to any farm
animals, in particular sheep, cattle and poultry. In addition, the
feeds or supplements may be given to farmed marine organisms such
as fish and shell fish. The composition may thus include one or
more feed substances or ingredients for such an animal.
[0087] The oil of the invention may be sold directly as oil and
contained in appropriate packaging, typically one piece aluminium
bottles internally coated with epoxy phenolic lacquer, and flushed
with nitrogen. The oil may contain one or more antioxidants (e.g.
tocopherol, vitamin E, palmitate) each for example at a
concentration of from 50 to 800 ppm, such as 100 to 700 ppm.
[0088] Suitable compositions can include pharmaceutical or
veterinary compositions, e.g. to be taken orally or cosmetic
compositions. The oil may be taken as such, or it may be
encapsulated, for example in a shell, and may thus be in the form
of capsules. The shell or capsules may comprise gelatine and/or
glycerol. The composition may contain other ingredients, for
example flavourings (e.g. lemon or lime flavour) or a
pharmaceutically or veterinary acceptable carrier or excipient.
[0089] Preferred features and characteristics of one aspect of the
invention are equally applicable to another aspect mutatis
mutandis.
[0090] The following Examples are provided to merely illustrate the
invention, and are not to be construed to be limiting.
COMPARATIVE EXAMPLES 1 AND 2 AND EXAMPLES 3 AND 4
Production of Arachidonic Acid (ARA)
[0091] One 1 ml vial of suspension Mortierella alpina strain CBS
168.95 (deposited by DSM N.V., 2600 MA Delft, The Netherlands (who
has authorized the present applicant to refer to this deposited
biological material) at Centraal Bureau voor Schimmelcultures
(CBS), P.O. Box 85167, 3508 AD Utrecht, The Netherlands, on 20 Feb.
1995 under deposit no. DS 30340) was stored at -80.degree. C. and
opened aseptically. The contents were used to inoculate a 500 ml
flask with 100 ml of a medium containing (g/l): [0092] glucose, 20;
[0093] yeast extract (Gistex.RTM. paste (solids 80%, protein
(Nx6.25) 46%, NaCl 16%, pH (2% solution) 5.6, ash 22%, total plate
count 10.sup.4/g, Enterobacteriaceae <10/g, E. coli <1/g,
yeast and moulds <100/g, available from DSM N.V., Savory
Ingredients PO Box 1, 2600 MA Delft), 12.5; [0094] antifoam
(Basildon 86/013K silicon/non-silicone antifoam compound used
according to manufacturer's instructions, Basildon Chemical
Company, Kimber Road, Abingdon, Oxford, England OX14 IRZ), 0.2.
[0095] The pH of the medium was adjusted to 7.0 before
autoclaving.
[0096] The culture was grown at 25.degree. C. for 48 hrs with
shaking at 250 rpm and used for the inoculation of four 2000
ml-flasks with 500 ml of a medium containing (g/l): glucose, 20;
yeast extract (Gistex.RTM. paste), 25; antifoam (Basildon 86/013K),
0.2. The pH before sterilization was 7.0.
[0097] These cultures were grown at 25.degree. C. for 24 hrs and
used for seeding a 5 m.sup.3 inoculation fermentor containing 2400
litres medium of the same composition as used in the 2000 ml-flasks
(the pH before sterilization was 6.0).
[0098] The fermentation temperature was set at 25.degree. C.,
agitation at 150 rpm, vessel pressure at 0.5 bar and aeration rate
at 0.5 VVM.
[0099] The culture from the inoculum fermenter was transferred to
the main fermenter after approx. 36 hours (the oxygen uptake rate
was >3 mmol/kg/h).
[0100] The main fermentor contained (g/l):
[0101] glucose, 35;
[0102] yeast extract (Expresa 2200.RTM. powder, low sodium brewer's
yeast, peptone (extract), solids>96%, total N>10%, amino N
6-7%, NaCl<1%, pH (2% solution 5.3-6.3), ash<12.5%,
homogenous powder available from DSM N.V., Savory Ingredients),
5.0;
[0103] NaH.sub.2PO.sub.4.2H.sub.2O, 1.0;
[0104] KH.sub.2PO.sub.4, 2.0;
[0105] MgSO.sub.4.7H.sub.2O 0.5;
[0106] Basildon 86/013K, 0.3; citric acid. 1H.sub.2O, 0.6;
[0107] ZnCl.sub.2, 0.010;
[0108] Fe.sub.2(SO.sub.4).sub.3 20% H.sub.2O, 0.025;
[0109] MnSO.sub.4.1H.sub.2O, 0.010; (pH before sterilization
5.0).
The glucose was sterilized separately and added to the main
fermentor after sterilization.
[0110] The fermentation lasted for 175 hours. The pH of the medium
was continued at about pH 6 (+/-0.1) with an aeration (air flow) of
0.5 VVM, the air pressure at 0.8 bar and agitation at 70 rpm.
Oxygen level was maintained at D.O..gtoreq.30% by sequentially
increasing agitation speed to 100 rpm and airflow to 0.9 VVM.
[0111] A sterile glucose solution of about 50% (w/w) was fed to the
fermentor to maintain the glucose concentration to above 10 g/l and
from about 30 to 78 hrs 625 kg of a 25% yeast extract solution was
fed to the fermentor with a feed rate controlled in such a way that
the ammonia concentration was <30 mg/l.
[0112] The experiment was performed four times (Examples 1 to 4),
and the glucose concentration of the culture medium was monitored
over time. A graph of glucose concentration, in g/kg, is shown in
FIG. 1, against the number of hours into fermentation. This shows
the last value of Example 4 was 2.2 g/kg at 172 hours. This was
three hours before the end of fermentation (EoF). The glucose level
was zero at about one hour before EoF.
[0113] In Comparative Examples 1 and 2 the concentration of the
carbon source (glucose) was well above 5 g/kg at the end of
fermentation. Indeed, at 10 hours before EoF, the glucose
concentration was about 20 g/kg. Thus, in Examples 1 and 2, the
concentration of glucose was such that it was not limiting on the
group of the micro-organisms, or on the production of ARA.
[0114] In Examples 3 and 4 the glucose concentration in the last
stage of fermentation, immediately preceding EoF, was controlled in
such a way in that about 10 hours before the EoF the concentration
of glucose was about 5 g/kg. During this last stage, over 10 hours,
the glucose was added at an addition rate of 0.5 g/kg/hr. The
glucose concentration was virtually zero at EoF. During this period
the consumption rate of the glucose was about twice the rate of
addition, namely about 1 g/kg/hr.
[0115] The concentration of glucose (g/kg) in the culture medium
over time, during fermentation is shown in Tables 1 to 4 (which
correspond to Examples 1 to 4).
TABLE-US-00001 TABLE 1 Time Glucose concentration (hrs) g/kg 0 48.7
24 57.6 28 47.8 54 46.4 78 62.0 102 62.5 126 48.2 150 42.5 167
20
TABLE-US-00002 TABLE 2 Time Glucose concentration (hrs) g/kg 0 47.8
28 32.5 54 32.2 78 41.3 102 49.5 126 46.8 150 29.9 165 17.1
TABLE-US-00003 TABLE 3 Time Glucose concentration (hrs) g/kg 0 49.2
28 60.1 54 40.8 78 34.0 102 37.3 126 23.2 150 16.7 172 7
TABLE-US-00004 TABLE 4 Time Glucose concentration (hrs) g/kg 0 45
28 34.1 54 34.7 78 29.2 102 38.1 126 45 150 23.5 172 2.2
[0116] At the end of fermentation, the micro-organisms and
surrounding aqueous liquid (the fermentation broth) was removed
from the fermenter. The broth underwent solid liquid separation to
remove some of the water. The remaining cells were then extruded,
and subjected to solvent-extraction using hexane. An ARA containing
microbial oil (hexane extractable lipids) was thus obtained from
cells undergoing each of the four different fermentation
protocols.
[0117] The percentage ARA content of the oil (on a weight by weight
basis) was then determined using the well-known FAME analytical
protocol (as detailed in AOCS Ce1b89). In Example 3 the
concentration of ARA in the microbial oil was 508 g/kg (50.8%). The
equivalent figure for Example 4 was 545 g/kg (54.5% ARA). By
comparison, the microbial oil extracted from the cells in
Comparative Examples 1 and 2 was much lower at 36.8% and 36.7%,
respectively.
* * * * *