US010633678B2

(12) United States Patent ( 10 ) Patent No.: US 10,633,678 B2

Streekstra et al. (45 ) Date of Patent: * Apr. 28, 2020
( 54 ) PREPARATION OF MICROBIAL OIL 1/005 ( 2013.01 ); C12P 7/6427 (2013.01 );
C12P 7/6481 (2013.01) ; A61K 2800/85
(71 ) Applicant: DSM IP Assets B.V., Te Heerlen (NL ) ( 2013.01)
(58) Field of Classification Search
(72) Inventors: Hugo Streekstra , Amsterdam (NL ); None
Petrus Joseph Maria Brocken , De See application file for complete search history.
Lier (NL )
(56 ) References Cited
(73 ) Assignee: DSM IP ASSETS B.V., Te Heerlen U.S. PATENT DOCUMENTS
(NL )
5,128,250 A 7/1992 Akimoto et al.
( * ) Notice : Subject to any disclaimer, the term of this 5,322,780 A 6/1994 Kawashima et al.
patent is extended or adjusted under 35 5,882,703 A 3/1999 Barclay
U.S.C. 154 (b ) by 327 days. 6,746,857 B2 6/2004 Higashiyama et al.
7,470,527 B2 12/2008 Streekstra et al.
This patent is subject to a terminal dis 2002/0037561 A1 * 3/2002 Barclay A61K 35/68
claimer. 435/134
2005/0202148 Al 9/2005 Streekstra et al.
( 21 ) Appl. No .: 14 /027,588 2009/0053342 A1 2/2009 Streekstra et al.

(22) Filed : Sep. 16 , 2013 FOREIGN PATENT DOCUMENTS

CN 1362522 A 12/2001
(65) Prior Publication Data EP 0223960 A2 6/1987
US 2014/0017742 A1 Jan. 16 , 2014 EP 1 035 211 9/2000
JP 64-38007 2/1989
JP 2-13388 1/1990
Related U.S. Application Data JP 02013388 1/1990
(60 ) Continuation of application No. 12/242,997 , filed on JP 2-142486 5/1990
JP 02142486 5/1990
Oct. 1, 2008, now abandoned , which is a division of JP 10-512444 12/1998
application No. 10 /518,949, filed as application No. WO WO 92/13086 8/1992
PCT/EP03/ 06552 on Jun . 20 , 2003, now Pat. No. (Continued )
7,470,527 .
( 30 ) Foreign Application Priority Data OTHER PUBLICATIONS
Hempenius et al., Food and Chemical Toxicology , 35 : 573-581,
Jun . 19 , 2002 ( EP ) 02254262 1997. *
Dec. 18 , 2002 (EP ) 02258713 (Continued )
Feb. 26 , 2003 (EP ) 03251169
Primary Examiner Allison M Fox
(51) Int. Cl. Assistant Examiner Qing Xu
C12P 7/64 ( 2006.01) (74 ) Attorney, Agent, or Firm -Shannon McGarrah ; Xi
A23L 33/12 (2016.01) Chen
A23K 20/158 (2016.01)
A23D 9/00 (2006.01) (57) ABSTRACT
A23L 33/115 (2016.01) The present invention provides a process for the production
C12N 1/00 (2006.01)
A61Q 19/00 (2006.01 ) of a microbial oil comprising culturing a micro -organism in
A61K 8/92 (2006.01) a two stage fermentation process where, in a last stage that
A61K 8/67 ( 2006.01 ) precedes the end of fermentation , the carbon source is:
CIB 1/10 ( 2006.01) consumed by the micro -organisms at a rate greater than it is
A23D 9/02 ( 2006.01 ) added to the medium ; added at a rate # 0.30 M carbon /kg
A61K 8/99 (2017.01)
medium ; or is rate limiting on the growth of the micro
organism . The micro -organisms thus have the carbon source
(52 ) U.S. CI. restricted so that they preferentially metabolise fats or lipids
CPC C12P 776472 ( 2013.01) ; A23D 9/00 other than arachidonic acid (ARA ), so increasing the pro
(2013.01); A23D 9/02 (2013.01) ; A23K 20/158 portion of ARA in the cells. A microbial oil is then recovered
( 2016.05); A23L 33/115 (2016.08 ); A23L from the micro - organism , using hexane as a solvent, that has
33/12 (2016.08 ); A61K 8/67 (2013.01); ACIK at least 50 % ARA and at least 90 % triglycerides.
8/925 (2013.01) ; A61K 8/99 (2013.01); A61Q
19/00 ( 2013.01) ; CIIB 1/10 ( 2013.01) ; C12N 18 Claims, No Drawings
 US 10,633,678 B2
Page 2

( 56 ) References Cited Kyle and C. Ratledge ), American Oil Chemists ' Society , Cham
paign , Illinois ( 1992), pp . 52-60 .
FOREIGN PATENT DOCUMENTS Totani et al, Lipids ( 1987 ) 22 ( 12 ): 1060-1062 .
Yamada et al, “ Production of Dihomo-Gamma - Linolenic Acid ,
WO WO 96/21037 7/1996 Arachidonic Acid and Eicosapentaenoic Acid by Filamentous Fungi,"
WO WO1996021037 7/1996 in Industrial Applications of Single Cell Oils ( ed . by D.J. Kyle and
WO WO 97/37032 10/1997 C.Ratledge ), American Oil Chemists' Society, Champaign , Illinois
WO WO 02/10322 2/2002 ( 1992 ), pp . 118-138 .
Shinmen et al, “ Production of arachidonic acid by Mortierella
OTHER PUBLICATIONS fungi”, Applied Microbiology and Biotechnology ( 1989 ) 31:11-16 .
Shimizu et al, “ Production of Eicosapentaenoic Acid by Mortierella
Eroshin et al, “ Arachidonic acid production by Mortierella alpina Fungi” , JAOCS , 65 (9 ): 1455-1459 , 1988 .
with growth -coupled lipid synthesis ” , Process Biochemistry ( 2000 ) Eroshin et al, “ Arachidonic acid production by Mortierella alpina
35: 1171-1175 . with growth - coupled lipid synthesis ”, Process Biochemistry, 35: 1171
International Search Report for PCT/EP03 /06552 , dated Mar. 9 , 1175 , 2000 .
2005, 5 pages. Shinmen et al, “ Production of arachidonic acid by Mortierella
Jang Hung -Der et al, “ Polyunsaturated fatty acid production with fungi” , Appl Microbiol. Biotechnol. 31 :11-16 , 1989 .
Mortierella alpina by solid substrate fermentation " , Botanical Bul Higashiyama et al, “ Effects of Mineral Addition on the Growth
letin of Academia Sinica (2000 ) 41( 1):41-48 . Morphology of and Arachidonic Acid production by Mortierella
Lindberg et al, “ Effect of temperature and glucose supply on the alpina 1 S - 4” , JAOCS 75, 1815-1819 ( 1998 ).
production of polyunsaturated fatty acids by the fungusMortierella Higaghiyama et al., Production of Arachidonic acid by Mortierella
alpina CBS 343.66 in fermentor cultures”, Applied Microbiology Fungi, Biotechnol. Bioprocess Eng., 2002 , 252-262, 7 .
and Biotechnology ( 1993 ) 39 :450-455 . Higashiyama et al., Enhancement of Arachidonic Acid Production
Shimizu , “ Production of C20 Polyunsaturated Fatty Acids by Micro by Mortierella alpina 1S - 4 , JAOCS, 1998 , 1501-1505, 75 ( 11).
bial Processes” , Oils- Fats -Lipids 1995 , Proc. World Congr. Int. Soc . Totani et al., The Filamentous Fungus Mortierella alpina , High in
Fat Res., 21st (1996 ),Meeting Date 1995, vol. 1 pp. 103-109 . Arachidonic Acid , Lipids, 1987 , 1060-1062, 22 ( 12 ).
Totani et al,“ IndustrialProduction of Arachidonic Acid by Mortierella” ,
Chapter 4 in Industrial Applications of Single Cell Oils (ed .by D.J. * cited by examiner
 US 10,633,678 B2
1 2
PREPARATION OF MICROBIAL OIL fermentation temperature which means the fermentation is
considerably slowed . The ARA content here is based on
RELATED APPLICATIONS extraction with a chloroform /methanol solvent mixture .
Another document in the ARA production field is WO
This is a continuation of application Ser. No. 12 /242,997 5 96/21037 .
(pending), filed Oct. 1, 2008 (published as US2009-0053342 EP - A - 1035211 ( Suntory ) describes a process for produc
A1), which is a divisional of application Ser. No. 10/518 , ing ARA and DHGLA lipids from M. alpina . However, the
949 , filed Dec. 17 , 2004 (issued as U.S. Pat. No. 7,470,527 ), calculation of ARA content is based either on the biomass
which is a U.S. nationalphase of PCT/EP 2003/006552, filed 10 (rather than an oil extracted from it), or results from an
Jun . 20 , 2003 , which claims benefit of European Applica analyticalmethod where the polyunsaturated fatty acids are
tions 03251169.3 , filed Feb. 26 , 2003, 02258713.3 , filed esterified first, and then extracted using a solvent (rather than
Dec. 18 , 2002 and 02254262.5 , filed Jun . 19 , 2002, the entire being extracted first, to produce an oil, and the ARA content
contents of each of which is hereby incorporated by refer then being determined on the basis of this oil).
ence in this application . 15 One report of a higher yield of ARA alleges a concentra
FIELD OF THE INVENTION tion in harvested mycelia ofnearly 70 % using the strain 15-4
of M. alpina (Shimizu S., Oils-Fats -Lipids 1995 , Proc .
The present invention relates to a process for the produc World Congr. Int. Soc . Fat Res., 21st ( 1996 ), Meeting Date
tion of a PUFA , optionally in a microbial oil, comprising 1995 , Volume 1 , pages 103-109 and Biochemical and Bio
culturing a micro -organism in a two-stage process and 20 physical Research communications, Vol. 150 (1 ), 1988 ,
subsequently recovering the microbial oil from the micro pages 335-441). However this percentage is based on the
organisms. The invention also relates to a novel ( e.g. micro cells, and is not the same as the ARA percentage in a
bial) oil resulting from such a process. In the oil 50 % or microbial oil . In fact the oil made only gave an ARA content
more of the lipids (or PUFAs, such as in the oil) are of 39.0 % ( Table 27.2 , page 105 ). (Note that the art uses a
arachidonic acid (ARA). The oil may have a low peroxide 25 variety of different ways to measure ARA content, which
value (POV ), of below 2.5 or 2.0 and /or a low anisidine may not necessarily be the same unit or based on the same
value (AnV), below 1.0 . The present invention also relates analytical protocol as the FIGURES quoted later in this
to foodstuffs and food supplements comprising, or generated specification ). Furthermore, this was only obtained when the
using, the microbial oil of the invention . M. alpina cells were allowed to stand at room temperature
30
INTRODUCTION for a further 6 days after fermentation , which is clearly not
a viable option for industrial production processes.
Polyunsaturated fatty acids, or PUFAs, are found natu There is, therefore, a need to identify ways to increase the
rally . A wide variety of different PUFAs are produced by bial oils and( and
proportion
in
so yield ) of arachidonic acid in the micro
particular in a way that can be employed on
different single cell organisms (algae , fungi , etc ). One par- 35
ticularly important PUFA is arachidonic acid (ARA ), one of an industrial scale .
a number of Long Chain Poly -Unsaturated Fatty Acids SUMMARY OF THE INVENTION
(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. 40 The present invention provides novel processes to pro
Arachidonic acid is a major precursor of a wide variety of duce a (microbial) oil with an increased proportion of
biologically active compounds, known collectively as eico arachidonic acid . This means arachidonic acid may be
sanoids, a group comprising prostaglandins, thromboxanes produced at a reduced cost and increased rate . In addition , as
and leukotrienes . Arachidonic acid is also one of the com the present invention does not rely on the genetic modifi
ponents of the lipid fraction of human breast milk and is 45 cation of micro -organisms involved in order to enhance
thought to be essential for optimal neurological development arachidonic acid production , the invention can help to meet
in infants. Arachidonic acid has a wide variety of different the increasing demand for natural, non -genetically modified
applications including use in infant formula , foodstuffs and food ingredients. In addition the oil has low oxidation
animal feeds. potential and so is suitable for incorporation into human
Arachidonic acid can be produced usingmicro -organisms 50 foods for which toxicity is particularly important, such as
and in particular using the filamentous fungi Mortierella . infant formula .
However, the percentage of arachidonic acid in the micro Accordingly, a first aspect of the invention relates to a
bial oil produced is usually too low . A number of attempts process for the production of a microbial oil or a polyun
have been made to try and increase the yield of arachidonic saturated fatty acid (PUFA ). The process comprises ferment
acid from Mortierella , but with varying degrees of success. 55 ing (or culturing ) a micro -organism inside a fermentation
Many of the attempts to increase arachidonic acid levels vessel, suitably in a culture medium , whereby before ( or at
have involved steps that cannot be readily used in an a stage which precedes) the end of fermentation ;
industrial setting. a ) the carbon source is consumed by the micro -organisms
For example, Eroshin et al , Process Biochemistry : (35 ) at a rate greater than it is added to the medium ;
2000 , pp 1171-1175 leaves the culture to sit for a period of 60 b ) the carbon source is added at a rate of # 0.30M
about a week after the end of fermentation . The amounts of carbon /kg medium per hour ;
ARA quoted are based on the biomass (and not on oil c ) the carbon source is rate limiting on the growth of the
extracted from it ) as this document does not describe the micro -organisms, or is restricted such that the micro
extraction of any oil. Totani et al, Industrial Applications of organismsmetabolise ( its own ) fat(s ) and /or lipid (s );
single cell oils , American Oil Chemists' Society Campaign , 65 d ) the rate of addition of the carbon source is reduced to ,
1992 , Chapter 4 , pp 52-60 and Lipids, vol. 22 No. 12 (1987 ) or is below the, rate of consumption of the carbon
pages 1060-1062 advocates the use of an unusually low source by the micro - organisms;
 US 10,633,678 B2
3 4
e ) the carbon source has been all used , or has a concen y - linolenic acid (GLA , 18 :3 026 );
tration in themedium of about zero , at or before the end a -linolenic acid (ALA , 18 : 3 23 ) ;
of fermentation ; conjugated linoleic acid (octadecadienoic acid , CLA );
f) the carbon source addition is stopped but fermentation dihomo -y- linolenic acid (DGLA , 20 :3 126 );
is allowed to continue ; and /or 5
arachidonic acid (ARA , 20 :4 26 ); and
g) the micro - organisms are subjected to conditions such eicosapentaenoic acid ( EPA , 20 :5 223 ).
that they metabolise fat( s) (e.g. inside the cell , such as Preferred PUFAs include arachidonic acid (ARA ),
a PUFA ) other than ARA in preference to ARA . docosohexaenoic acid (DHA), eicosapentaenoic acid ( EPA )
A second aspect of the present invention relates to a and/or y- linoleic acid (GLA ). In particular, ARA is preferred .
microbial oil which comprises at least 50 % ( or at least 50.5 , 10
51 or 52 % ) arachidonic acid (ARA ). It may have up to 55 , Fermentation
57 or 60 % ARA . This oil may have :
a ) a triglyceride content of at least 90 % ; The fermentation /culturing will typically be carried out in
b ) a POV of less than 2.5 ; a suitable fermenter (or fermentation vessel) containing a
c ) an AnV of less than 1.0 ; and / or 15 (liquid , usually aqueous) culture medium . A main fermenter
d ) a phospholipid content below 5 % . vessel will normally be aseptically inoculated from a small
The oil can be preparable by the process of the first aspect. feed fermenter. Typically a submerged and /or aerobic fer
It may be hexane extracted . mentation process is employed . This may take place in a
It is thought that once the concentration of carbon source deep tank fermenter. The fermenter may be equipped with
is reduced or restricted, the cells startmetabolising the fats 20 devices to monitor and/or change pH and temperature. The
or lipids inside the cell . However , the cells consume fats vessel may additionally be adapted to perform , or allow to
other than ARA first . In this way the proportion of ARA in be conducted , aeration and/or mixing of the cells and liquid ,
the fats or lipids in the cells increases. Hence the process of such as agitation of the solution . This may be stirring, for
the first aspect can , in this way, lead to a higher ARA content 25 example achieved using mechanical means .
oil of the second aspect . Suitably the volume of the fermenter is at least 10 , 20 , 40
or even 60 m3. Volumes as high as 100 or even 150 m² can
DETAILED DESCRIPTION OF THE be used .
INVENTION The fermentation will typically last for 10 days or less ,
preferably 9 or less days, more preferably 8 or less days . It
Micro -Organisms 30 may be at least 4 , 5 , 6 or 7 days .
Optionally the fermentation may be for 150 to 200 hours ,
The micro -organism employed may be a bacteria , yeast , such as 160 to 190 hours, eg . from 170 to 180 hours. The end
algae or fungus. Preferably a fungus is used and in particular of fermentation is usually the point atwhich agitation and /or
a filamentous fungus is used . Preferred fungi are of the order aeration is stopped . This can be when the fermenter vessel,
Mucorales. The fungus may be of the genus Mortierella, 35 and /or ancillary equipment, is (effectively ) switched off. The
Phycomyces, Entomophthora , Pythium , Thraustochytrium , micro - organismsmay then be removed from the fermenter.
Blakeslea , Rhizomucor or Aspergillus. Preferred fungi are of The fermentation may be at a temperature of from 20 to
the species Mortierella alpina . Preferred yeasts are of the 40 ° C.
genus Pichia or Saccharomyces, for example Pichia ciferrii.
Bacteria can be of the genus Propionibacterium . Suitable 40 Carbon and Nitrogen Sources
algae arc dinoflagellate and /or belong to the genus Crypth
ecodinium , Porphyridium or Nitschia , for example are of the Any suitable medium may be used in the fermentation , for
species Crypthecodinium cohnii. example a medium appropriate to the micro -organism being
The micro -organism strains used in the present invention used . The carbon source can comprise ( complex sources
may be a naturally occurring or commonly used industrial 45 such as ) maltodextrin , oat flour, oat meal, molasses, veg
strain . The strain may not have been genetically altered for etable (e.g. soy bean ) oil, malt extract , starch , ethanol or soy
example , it may not be transformed with a vector nor may bean oil. Preferred (non -complex ) carbon sources include
it contain heterologous gene (s).Given the currentpreference carbohydrates or sugars , such as fructose, maltose , sucrose ,
in some quarters for foodstuffs which do not contain geneti xylose, mannitol, glucose or lactose or glycerine (e.g. from
cally engineered ingredients , the micro -organism employed 50 a vegetable source), citrate, acetate, glycerol, ethanol or ( e.g.
may be a strain which has not been so modified . sodium ) ascorbate . In a preferred embodiment of the inven
tion the carbon source is or comprises glucose , and in
Polyunsaturated Fatty Acids (PUPAS ) particular is glucose syrup .
Suitable nitrogen sources include yeast extract , urea and
The PUFA can either be a single PUFA or two or more 55 peptone. The medium can exclude agar.
different PUFAS. Preferred nitrogen and /or carbon sources are water
The or each PUFA can be of the n -3 or n -6 family. soluble or water miscible .
Preferably it is a C18 , C20 or C22 PUFA . Itmay be a PUFA Individual components of the medium (such as the nitro
with at least 18 carbon atoms and /or 3 or 4 double bonds . gen and /or carbon sources )may either (all) be present at the
The PUFA (s ) can be isolated in the form of a free fatty acid , 60 start of fermentation , added continuously during fermenta
a salt , as a fatty acid ester (e.g. methyl or ethyl ester ), as a tion or added in stepwise or batches. In particular, the
phospholipid and /or in the form of a mono-, di- or triglyc amount of carbon source present in the medium will typi
eride . cally be controlled as described below , preferably by con
Suitable (n -3 and n -6 ) PUFAs include : trolling the rate of addition of the carbon source .
docosahexaenoic acid (DHA , 22 :6 E23 ), suitably from 65 The nitrogen and /or carbon sources can be supplied (or
algae or fungi , such as the (dinoflagellate ) Cryptheco added ) separately , or supplied simultaneously , or supplied as
dinium or the (fungus) Thraustochytrium ; a combined preparation . They may thus present in the same
 US 10,633,678 B2
5 6
composition ( if thought necessary ) which is preferably a may exceed the rate of its consumption by the micro
liquid . The carbon and /or nitrogen sources can be added (to organisms. In the second or last stage of fermentation the
the fermenter vessel) either before the fungal cells are added amount of the carbon source being added can be decreased
(to the vessel), in other words prior to inoculation , or during source or stopped altogether. This means that the amount of carbon
fermentation alternatively they may be added both before 5 the second available to the micro - organism will decrease during
fermentation and during. or last stage of fermentation . Typically in a
second , final or last stage , or towards the end of fermenta
Culture Medium tion , the carbon source can be :
consumed by the micro -organisms at a rate greater than it
is added to the medium (for example the rate of
The culturemedium is preferably an aqueous liquid . This 10 addition is less than the rate of consumption );
may additionally contain other substances to assist in the added at a rate # 0.30 M carbon /kg medium per hour, such
fermentation , for example a chelating agent (e.g. citric acid ), as # 0.25 or # 0.20 , but at least 0.01, 0.02 or 0.05 M
an anti-foaming agent (e.g. soy bean oil ), a vitamin ( e.g. carbon /kg medium /hr ( the units here referring to the
thiamine and /or riboflavin ), any necessary catalytic metals moles, or molar amount, of carbon in the carbon source ,
( for example, alkali earth metals such as magnesium or 15 rather than the weight or moles of the carbon source
calcium , or zinc or iron and /or other metals such as cobalt itself);
and copper), phosphorus (e.g. phosphate ) and /or sulphur rate limiting on the growth and/or (PUFA ) production of
(e.g. sulphate ). The medium may, if necessary, contain an the micro -organisms.
additive oil such as olive or soybean oil ,however preferably Typically , the concentration of the carbon source during
the medium does not contain such an oil. 20 the second stage is # 10 g carbon source/kg of medium ,
The (optimum ) growth (or fermentation ) temperature may preferably from 0.01 or 0.1 to 8 or 10 g/kg ,more preferably
vary depending on the micro -organism used . However, it is from 0.5 to 5 g /kg and even more preferably from 1 or 2 to
preferably from 20 to 40 ° C. and more preferably from 22 4 or 5 g /kg . This means hat, on average during the last stage
to 30 or 32° C . In particular the temperature the fermentation of fermentation, there will be # 0.30M carbon per kg of
is carried out at will be 222 ° C.or < 25 ° C., eg . 22 to 30 ° C., 25 medium , preferably from 0.003 M to 0.3M carbon per kg .
such as from 23-28 ° C. The pH of the aqueous liquid during Advantageously this is from 0.015M to 0.17M carbon per kg
fermentation may be from 4 to 10 , such as from 5 to 8 , and even more preferably from 0.03M to 0.17M carbon per
optimally from 6 to 7 . kg.
The medium will typically be stirred or agitated during When the carbon source comprises glucose , typically the
fermentation to help facilitate aeration . The aqueous liquid 30 concentration of glucose in the last stage ) will be on
and the cells are suitably eithermixed or agitated . This may average # 10 g/kg ofmedium , preferably from 0.01 or 0.1 to
be achieved if aeration is provided , such as by bubbling a 8 or 10 g/kg. Advantageously this is from 0.5 to 5 g /kg and
gas, e.g. air, into the aqueous liquid . This may serve the even more preferably from 1 or 2 to 4 or 5 g/kg medium . In
additional purpose of providing oxygen to the fungal cells : this sense medium includes the cells and the aqueous culture
hence the fermentation is preferably an aerobic one . Other 35 medium , that is to say it is the “ broth ” ( cells and surrounding
means of agitation or mixing include stirring, for example liquid ).
using an impeller. This may be of a hydrofoil axial flow The rate of addition of the carbon source in the last stage
design or may be designed so that the aqueous medium is is preferably no more than 0.03M carbon per kg , preferably
forced radially outwards from the impeller (such as a no more 0.025 or 0.02 M carbon per/kg (medium ). Prefer
turbine ). Even if there is no stirring it is preferred that the 40 ably the rate of addition is about 0.015M carbon per /kg . If
microbial cells are provided with oxygen during fermenta the carbon source is glucose, then preferably the rate of
tion , and so aeration (e.g. by bubbling air , oxygen or other addition of glucose is less than 1.0, for example less than
oxygen - containing gas) is advantageous here . Aeration may 0.8 , for example less than 0.5 g glucose per/kg medium per
be at from 0.1 to 2.0 , such as from 0.5 to 1.0 vvm . hour.
Preferably the volume of the fermenter is at least 2 or 5 45 Preferably the rate of addition of the carbon source in the
litres , preferably at least 10 litres. However, for fermenters last stage is approximately half that of the rate of consump
used in industry, or on an industrial scale , the vessel volume tion of the carbon source by the micro - organisms.However,
is preferably at least 50, 100, 500 or 1,000 litres . 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
Last (or Second ) Stage of Fermentation 50 to 2.2 . Alternatively , the rate of addition may be from
30-70 % , such as from 40 to 60 % , optimally from 45 to 55 % ,
The fermentation process may be split into at least two of the rate of consumption.
stages. A second or last stage , which may immediately The appropriate concentration of the carbon source during
precede the end of fermentation , can be characterised by a the second stage of the fermentation can be achieved by
decrease in the amount of the carbon source available to the 55 carefully controlling the rate of addition of the carbon
micro -organism or any of the features (a ) to (g ) as given in source . Typically , this will be decreased as appropriate
the first aspect . Typically, this stage can begin from 15 to 2 during , or to precipitate the onset of the last stage. Periodic
hours before the end of fermentation , preferably less than or sampling and analysis of the culture can be used to deter
at 10 hours from the end of fermentation , more preferably mine the concentration of the carbon source and to make
from 3 to 5 hours from the end of fermentation . Preferably, 60 adjustments as necessary to the rate ofaddition of the carbon
this stage will typically begin less than 10 days after the source . This may be done automatically using a computer
beginning of fermentation ,more preferably it will begin less system .
than 9 days after , even more preferably less than 8 days after.
During a first or earlier stage of fermentation the carbon Pasteurisation Process
source may be in excess . Thus the amount of carbon source 65
available may not be limiting to on the growth of the Pasteurisation will usually take place after fermentation
micro -organisms. The rate of addition of the carbon source has finished . In a preferred embodiment, pasteurisation will
 US 10,633,678 B2
7 8
finish the fermentation ,because the heat during pasteurisa processing , or it can be subjected to one or more refining
tion will kill the cells . Pasteurisation may therefore be steps. Suitable refining protocols are described in Interna
performed on the fermentation broth (or the cells in the tional patent application no. PCT/EP01/08902 (the contents
liquid (aqueous) medium ), although it can be performed on of this document and all others described herein arc hereby
the microbial biomass obtained from the broth . In the former 5 incorporated by reference). For example , the oil can be
case, pasteurisation can take place while the microbial cells subjected to acid treatment or degumming , alkali treatment
are still inside the fermenter. Pasteurisation preferably takes or free fatty acid removal, bleaching or pigment removal,
place before any further processing of the microbial cells , filtration, winterisation (or cooling, for example to remove
for example granulation (e.g. by extrusion ) crumbling, or 10 saturated triglycerides ), deodorising (or removal of free fatty
kneading .
Preferably the pasteurisation protocol is sufficient to acids ) and/or polishing (or removal of oil- insoluble sub
stances ).
inhibit or inactivate one or more enzymes that can adversely
affect or degrade a PUFA or microbial oil , for example a purposes, and canoilbeis added
The resulting particularly suitable for nutritional
to (human ) foods or (animal)
lipase . feedstuffs . Examples include milk , infant formula , health
Once fermentation has been finished , the fermentation 15 drinks, bread and animal feed .
broth may be filtered , or otherwise treated to remove water
or aqueous liquid . After water removal, one may obtain a Purification /Refinement
biomass " cake” . If pasteurisation has not taken place , then
the dewatered cells (or biomass cake) can be subjected to The microbial oil may be refined and purified . This may
pasteurisation 20
involve removing one or more of the following components:
Oil Extraction a phospholipid , trace metal, pigment, carbohydrate, protein ,
free fatty acid (FFA ), oil insoluble substance , water
If desirable , and for example after fermentation is fin insoluble substance , soap or saponified substance , oxidation
ished , the micro -organisms may be killed or pasteurised . 25 product, sulphur,mono- or diglyceride, pigment decompo
This may be to inactivate any undesirable enzymes, for sition product, solvent and /or sterol. The purifying may
example enzymes that might degrade the oil or reduce the reduce or remove “off-flavours ” and /or improve the stability
yield of the PUFAs. of the oil.
After culturing or fermentation is complete or has ended , To effect this the process ( e.g. purifying ) may comprise
the fermentation broth (cells and aqueous liquid ) may then 30 degumming (or acid treatment), neutralization (or alkali
be removed from the fermenter, and if necessary liquid treatment), water washing, bleaching, filtering, deodorising ,
,
(usually water) removed therefrom . Any suitable solid liquid polishing and /or cooling (or winterization ). Preferably the
separation technique can be used . This (dewatering ) may be purifying comprises acid treatment and / or alkali treatment
by centrifugation and /or filtration . The cells may be washed , (degumming and neutralisation ). Alternatively purifying
for example using an aqueous solution (such as water) for 35 methods may comprise bleaching and/or deodorization .
example to remove any extracellular water-soluble or water Preferably however the purifying will involve bleaching
dispersible compounds. An oil can then be recovered from and /or deodorization , and optimally in addition acid and /or
the microbes, for example using a solvent so that the oilmay alkali treatment.
be solvent-extracted , preferably hexane-extracted .
The oil may have no (or be substantially free from ) GLA 40 Oils
and / or DGLA .
The second aspect of the present invention provides a
PUFA Extraction Process 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 %
The PUFA (or microbial oil , usually containing the 45 or more of this PUFA , such as ARA . It can have triglyceride
PUFA ) may be extracted from (e.g. dried ) granules (e.g. content of at least 90 % . Preferably the microbial oil com
extrudates ) containing the cells . The extraction can be prises from 50 , 55 or 60 to 90 % arachidonic acid , more
performed using a solvent. Preferably a non -polar solvent is preferably from 60 to 80 % and even more preferably from
used , for example a C1-8, e.g. C2-6 , alkane , for example 60 to 70 % arachidonic acid .
hexane . One may use carbon dioxide (in a liquid form , for 50 Preferably the microbial oil has a triglyceride content of
example in a super critical state ). from 90 to 100 % , such as at least 90 or 96 % , preferably at
The cells may thus be subjected to extraction , such as with least 98 % , more preferably at least 99 % and optimally above
an organic solvent, preferably under nitrogen flow . Other 99.5 % . Typically, the microbial oil will have an eicosapen
usable organic solvents include ether , methanol, ethanol, taenoic acid (EPA ) content of below 5 % , preferably below
chloroform , dichloromethane and /or petroleum ether . 55 1 % and more preferably below 0.5 % . The oil may have less
Extraction with methanoland petroleum ether and /or extrac than 5 % , less than 2 % , less than 1 % of each of C20, C20:3 ,
tion with a one - layer solvent system consisting of chloro C22 :0 and/or C24:0 polyunsaturated fatty acid (PUFAs). The
form ,methanol, and water can also be used . Evaporation of free fatty acid (FFA ) content may be # 0.4 , 0.2 or 0.1 % .
the organic solvent(s) from the extract under reduced pres Of the triglycerides, preferably at least 40 % , such as least
sure can give a microbial oil containing arachidonic acid at 60 50 % , and more preferably at least 60 % of the PUFAs present
a high concentration . are at the C -position of the glycerol (present in the triglyc
Preferably, the solvent is allowed to percolate over the eride backbone ) also known at the 1 or 3 position . It is
dried granules. Suitable micro -organism granulation and preferred that at least 20 % , such as least 30 % , more pref
extrusion techniques and subsequent extraction of a micro erably at least 40 % of the PUFA (s ) is at the B (2 ) position .
bial PUFA containing oil, are described in WO- A - 97/37032 . 65 The phospholipid content of the oil is suitably at a
The solvent allows one to obtain a crude PUFA containing maximum of 5 % , 3 % or 2 % and /or may be at a minimum of
oil. This oil can be used in that state , without further from 0.1 , 0.5 or 1.0 % .
 US 10,633,678 B2
9 10
Typically the microbial oil will be one obtainable by the aluminium bottles internally coated with epoxy phenolic
process of the first aspect invention . Preferably the oil will lacquer, and flushed with nitrogen . The oil may contain one
have been isolated from a fungus, more preferably the oil is or more antioxidants ( e.g. tocopherol, vitamin E , palmitate )
isolated from Mortierella and in particular from M. alpina . each for example at a concentration of from 50 to 800 ppm ,
The oil is suitably hexane extracted . 5 such as 100 to 700 ppm .
Suitable compositions can include pharmaceutical or vet
ARA Content erinary compositions, e.g. to be taken orally or cosmetic
compositions . The oil may be taken as such , or it may be
Purely for the sake of clarity , the calculation of the encapsulated , for example in a shell , and may thus be in the
percentage ARA content will be explained , especially as the 10 form of capsules. The shell or capsules may comprise
literature can , on occasions, calculate the ARA content on a gelatine and /or glycerol. The composition may contain other
different basis . The percentage of ARA is based on the oil ingredients, for example flavourings ( e.g. lemon or lime
(that has been extracted from the biomass ), and not the flavour ) or a pharmaceutically or veterinary acceptable
biomass itself . It is on a weight by weight basis. It is based 15 carrier or excipient.
on an oil extracted by hexane , and is therefore based on Preferred features and characteristics of one aspect of the
hexane extractable lipids (HEL ). It is based on the total invention are equally applicable to another aspect mutatis
amount of oil, and not on the total amount of fatty acids mutandis.
(which can sometimes give a misleading higher FIGURE ). The following Examples are provided to merely illustrate
The ARA content is determined by the well -known FAME the invention , and are not to be construed to be limiting.
analytical protocol ( using the fatty acid methyl esters ), 20 Comparative Examples 1 and 2 and Examples 3
detailed in AOCS Cel b89. Different solvents will extract
different lipids. Note that in the present case , the oil is first and 4
extracted with hexane, and then the ARA content of the oil
determined by FAME analysis. This will give a different Production of Arachidonic Acid (ARA )
result from first esterifying the arachidonic acid (e.g. while 25 One 1 ml vial of suspension Mortierella alpina strain CBS
still in the cells) and then extracting the resulting methyl
esters for further analysis . 168.95 (deposited by DSM N.V., 2600 MA Delft , The
Netherlands (who has authorized the present applicant to
Peroxide Value (POV ) refer to this deposited biological material) at Centraal
30 Bureau voor Schimmelcultures (CBS), P.O. Box 85167,
Preferably the POV of the microbial oil is no more than 3508 AD Utrecht, The Netherlands, on 20 Feb. 1995 under
3.0 , 2.5 or 2.0 . However, much lower POV values can be deposit no . DS 30340 ) was stored at -80 ° C. and opened
obtained using the process of invention , and these values aseptically . The contents were used to inoculate a 500 ml
may be less than 1.5 or less than 1.0 . Values less than 0.8 and 35 flaskglucose
with , 100
20 :
ml of a medium containing (g /1):
even less than 0.4 can be obtained .
yeast extract (Gistex® paste ( solids 80 % , protein
Anisidine Value (AnV ) (Nx6.25 ) 46 % , NaCl 16 % , pH (2 % solution ) 5.6 , ash
22 % , total plate count 104/g , Enterobacteriaceae< 10 /g ,
Preferably the anisidine value of the microbial oil is no E. coli < 1/ g , yeast and moulds< 100 / g , available from
more than 1.0 , for example no more than 0.6,0.3 or even no 40 DSM N.V., Savory Ingredients PO Box 1, 2600 MA
more than 0.1 . Delft ), 12.5 ;
antifoam ( Basildon 86 / 013K silicon /non -silicone anti
Uses and Products foam compound used according to manufacturer's
instructions, Basildon Chemical Company, Kimber
A third aspect of the invention relates to a composition 45 Road , Abingdon , Oxford , England OX14 IRZ ), 0.2 .
comprising the oil of the second aspect, and where appro The pH of the medium was adjusted to 7.0 before auto
priate one or more (additional) substances . The composition claving
may be a foodstuff and /or a food supplement for animals or The culture was grown at 25 ° C. for 48 hrs with shaking
humans. In embodiments of the invention which are for at 250 rpm and used for the inoculation of four 2000
human consumption the oils may be rendered suitable for 50 ml- flasks with 500 ml of a medium containing (g/l): glucose ,
human consumption , typically by refining or purification of 20 ; yeast extract (Gistex® paste ), 25 ; antifoam (Basildon
the oil obtained from the microbes . 86 /013K ), 0.2 . The pH before sterilization was 7.0 .
The composition may be infant formula or (human ) These cultures were grown at 25 ° C. for 24 hrs and used
foodstuffs. Here the composition of the formula may be for seeding a 5 m inoculation fermentor containing 2400
adjusted so it has a similar amount of lipids or PUFAs to 55 litres medium of the same composition as used in the 2000
normal breast milk . Thismay involve blending themicrobial ml- flasks ( the pH before sterilization was 6.0 ).
oil of the invention with other oils in order to attain the The fermentation temperature was set at 25 ° C., agitation
appropriate composition . at 150 rpm , vessel pressure at 0.5 bar and aeration rate at 0.5
The composition may be an animal or marine feed com VVM .
position or supplement. Such feeds and supplements may be 60 The culture from the inoculum fermenter was transferred
given to any farm animals , in particular sheep, cattle and to the main fermenter after approx . 36 hours (the oxygen
poultry . In addition , the feeds or supplements may be given uptake rate was > 3 mmol/kg /h ).
to farmed marine organisms such as fish and shell fish . The The main fermentor contained ( g/ l):
composition may thus include one or more feed substances glucose, 35 ;
or ingredients for such an animal. 65 yeast extract (Expresa 2200® powder, low sodium brew
The oil of the invention may be sold directly as oil and er's yeast , peptone ( extract ), solids> 96 % , total N > 10 % ,
contained in appropriate packaging, typically one piece amino N 6-7 % , NaCl< 1 % , pH (2 % solution 5.3-6.3 ),
 US 10,633,678 B2
11 12
ash < 12.5 % , homogenous powder available from DSM TABLE 2
N.V., Savory Ingredients) , 5.0 ;
Glucose concentration
NaH2PO42H20 , 1.0 ; Time (hrs ) g /kg
KH PO4, 2.0 ; 5 0 47.8
MgSO4.7H2O 0.5 ; 28 32.5
Basildon 86 /013K , 0.3 ; citric acid . 1H20 , 0.6 ; 54
78
32.2
41.3
ZnCl2 , 0.010 ; 102 49.5
Fe2(SO4)3 20 % H20 , 0.025 ; 10
126
150
46.8
29.9
MnSO4.1H20 , 0.010 ; (pH before sterilization 5.0 ). 165 17.1
The glucose was sterilized separately and added to the main
fermentor after sterilization .
The fermentation lasted for 175 hours. The pH of the TABLE 3
medium was continued at about pH 6 (+/- 0.1 ) with an 15
aeration (air flow ) of 0.5 VVM , the air pressure at 0.8 bar Glucose concentration
and agitation at 70 rpm . Oxygen level was maintained at Time (hrs) g /kg
D.O.230 % by sequentially increasing agitation speed to 100 0 49.2
rpm and airflow to 0.9 VVM . 28 60.1
54 40.8
A sterile glucose solution of about 50 % (w /w ) was fed to 20 78 34.0
the fermentor to maintain the glucose concentration to above 102 37.3
10 g /l and from about 30 to 78 hrs 625 kg of a 25 % yeast 126 23.2
extract solution was fed to the fermentor with a feed rate 150
172
16.7
7
controlled in such a way that the ammonia concentration
was < 30 mg/1. 25
The experiment was performed four times (Examples 1 to
4 ), and the glucose concentration of the culturemedium was TABLE 4
monitored over time. A graph of glucose concentration , in Glucose concentration
g /kg , is shown in FIG . 1, against the number of hours into Time (hrs ) g /kg
fermentation . This shows the last value of Example 4 was 30
2.2 g /kg at 172 hours. This was three hours before the end 28
0 45
34.1
of fermentation ( EoF ). The glucose level was zero at about
one hour before EoF . 54 34.7
78 29.2
In Comparative Examples 1 and 2 the concentration of the 35 102
126
38.1
45
carbon source ( glucose ) was well above 5 g/kg at the end of 150 23.5
fermentation . Indeed , at 10 hours before EoF , the glucose 172 2.2
concentration was about 20 g/kg. Thus, in Examples 1 and
2 , the concentration of glucose was such that it was not At the end of fermentation , the micro - organisms and
limiting on the group of the micro -organisms, or on the 40 surrounding aqueous liquid ( the fermentation broth ) was
production of ARA .
In Examples 3 and 4 the glucose concentration in the last liquid separationtheto remove
removed from fermenter. The broth underwent solid
some of the water . The remain
stage of fermentation , immediately preceding EoF , was ing cells were then extruded , and subjected to solvent
controlled in such a way in that about 10 hours before the extraction using hexane . An ARA containing microbial oil
EoF the concentration of glucose was about 5 g /kg. During 45 (hexane extractable lipids) was thus obtained from cells
this last stage, over 10 hours , the glucose was added at an undergoing each of the four different fermentation protocols .
addition rate of 0.5 g/kg/hr. The glucose concentration was The percentage ARA content of the oil ( on a weight by
virtually zero at EoF. During this period the consumption weight basis ) was then determined using the well -known
rate of the glucose was about twice the rate of addition , FAME analytical protocol ( as detailed in AOCS Celb89 ). In
namely about 1 g /kg/hr. 50 Example 3 the concentration of ARA in themicrobial oil was
The concentration of glucose (g /kg) in the culture medium 508 g/kg (50.8 % ). The equivalent FIGURE for Example 4
over time, during fermentation is shown in Tables 1 to 4 was 545 g/kg (54.5 % ARA ). By comparison, the microbial
(which correspond to Examples 1 to 4 ). oil extracted from the cells in Comparative Examples 1 and
TABLE 1
2 was much lower at 36.8 % and 36.7 % , respectively.
55
Glucose concentration
We claim :
Time (hrs) g /kg 1. A fermentation process for production of a microbial oil
comprising at least 40 % arachidonic acid (ARA ), the pro
0
24
48.7
57.6
cess comprising culturing a microorganism which is Mor
28 47.8 60 tierella alpina in a two -stage process in a culture medium
54 46.4 inside a fermentation vessel, whereby at a second stage of
78
102
62.0
62.5
the fermentation process
126 48.2 (a ) a carbon source is rate limiting on the growth of the
150 42.5 microorganism ,
167 20 65 (b ) the carbon source is restricted such that the microor
ganism metabolises fat(s) and /or lipid (s ) in preference
to arachidonic acid (ARA ),
 US 10,633,678 B2
13 14
(c ) the carbon source is consumed by themicroorganism 8. The process according to claim 1 , wherein the oil
at a rate greater than it is added to the medium , comprises at least 50 % ARA (wt/wt) based on the oil.
(d ) the carbon source is added at an addition rate of 0.5 9. The process according to claim 1, wherein a concen
g /kg/hr, and tration of the carbon source in said second stage is < 4 g / kg
(e ) the carbon source is controlled so that the concentra 5 of medium .
tion of the carbon source is about 5 g/kg of medium 10. The process according to claim 1, wherein the solvent
is hexane .
about 10 hours before the end of fermentation ; 11. The process according to claim 8, wherein :
whereby the process further comprises extracting the oil concentration of the carbon source in the second stage is
from the micro -organism using a non -polar solvent, 10 on average s0.17 M carbon /kg medium .
and wherein the oil has a triglyceride contentof at least 12. The process according to claim 8 , wherein fermenta
90 % . tion is carried out at a temperature 222 ° C. and s30 ° C.
2. The process according to claim 1, wherein : 13. The process according to claim 1, wherein a concen
concentration of the carbon source in the second stage is tration of the carbon source in said second stage is < 4 g /kg
on average s0.17M carbon /kg medium . 15
of medium .
3. The process according to claim 1, wherein 14. The process according to claim 8, wherein the carbon
fermentation is carried out at a temperature 222 ° C. and source is glucose .
s30 ° C. 15. The process according to claim 8 , wherein during an
4. The process according to claim 1 , wherein the carbon earlier stage of fermentation , the carbon source is being
source is glucose . 20 added , wherein the earlier stage is prior to the second stage .
5. The process according to claim 1, wherein during an 16. The process according to claim 15 , wherein during
earlier stage of fermentation , the carbon source is being said earlier stage the carbon source is in excess.
added , wherein the earlier stage is prior to the second stage. 17. The process according to claim 8 , wherein the solvent
6. The process according to claim 5 , wherein during said is hexane.
earlier stage the carbon source is in excess . 25 18. The process according to claim 8, wherein cells from
7. The process according to claim 1, wherein cells of the the microorganism are pasteurized after fermentation is
microorganism are pasteurized after fermentation is fin finished .
ished .