The Journal of Supercritical Fluids 134 (2018) 260–268

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The Journal of Supercritical Fluids

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Perspectives on processing of high value lipids using supercritical fluids T

⁎
Owen Catchpole , Teresa Moreno, Fernando Montañes, Stephen Tallon
Callaghan Innovation, Integrated Bioactive Technologies, Industrial Research Limited, 69 Gracefield Road, 5040 Lower Hutt, New Zealand

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: Opinions regarding the state-of the-art, issues and future perspectives for the processing and production of high
Supercritical extraction value lipids using supercritical fluids are discussed including examples from New Zealand. The categories of high
Seed oils value lipids discussed are seed oils, marine neutral lipid extracts and polyunsaturated omega-3 fatty acid con-
Carotenoids centrates, carotenoid-rich extracts, and phospholipid-rich extracts. Commercial production of carotenoid-rich
Marine lipids
extracts has been a growth area, particularly astaxanthin-rich oleoresin produced from the micro-algae
PUFA concentrates
Phospholipids
Haematococcus pluvialis. The main research and development trends observed in the processing of feed streams to
produce these high value lipid products are the use of ultra-high pressures for CO2 extraction, the use of propane
and dimethyl ether as alternative extraction solvent, and semi-preparative supercritical chromatography to
produce omega-3 concentrates. New product opportunities include phospholipid concentrates from marine and
dairy materials, EPA and/or DHA-rich oils and concentrates from GM-modified seeds, and a broader range of
carotenoid-rich extracts obtained from microalgae.

1. Introduction glycolipids, ceramides, cerebrosides and gangliosides. There are four

Lipids are one of the three main macronutrients critical in the
human diet, the others being proteins and carbohydrates. There appears
to be no standard definition for a lipid, but common features of defi-
nitions include not water soluble, fat or oil-like, and of biological origin.
Lipids are further categorized as being either neutral lipids and complex
lipids. Neutral lipids are generally considered to encompass partial to
full glycerides, wax esters, free fatty acids, free fatty alcohols, car-
otenoids, sterols, tocopherol and lignin-type antioxidants and biologi-
cally-derived hydrocarbons. Complex lipids include phospholipids,
main classes of high value lipids that are discussed herein. These are:
high value seed oils; marine neutral lipid oils including omega-3 con-
centrates; carotenoid-rich oleoresins; and complex lipid-rich extracts.
The state of the art in supercritical processing of these high value lipids
was reviewed in 2009 [1]. Here, we present our opinions on the current
state of the art including some recent research and development activity
and commercial products, a discussion of the issues and gaps in
knowledge that are hindering further commercial exploitation; and
possible future perspectives. Each category of high value lipid is dis-
cussed with regard to these parameters. The possible future

⁎
Corresponding author.
E-mail address: Owen.Catchpole@callaghaninnovation.govt.nz (O. Catchpole).

https://doi.org/10.1016/j.supflu.2017.12.001
Received 13 October 2017; Received in revised form 1 December 2017; Accepted 1 December 2017
Available online 02 December 2017
0896-8446/ © 2017 Elsevier B.V. All rights reserved.
O. Catchpole et al.
perspectives are shown schematically in Fig. 1 with respect to current
and possible future extraction solvents, noting however that super-
critical CO2 is still the preferred solvent unless the alternative solvent
offers a substantial technical and economic benefit.
2. Current state and challenges
2.1. High value seed oils
The extraction of seed oils is one of the most extensively studied
areas in supercritical CO2 extraction. Initial research in the field was
focussed on supercritical CO2 as an alternative extraction technology to
solvent extraction using hexane or petroleum ether for large scale ex-
traction of commodity oils. Oil solubility in high pressure CO2 [2],
extraction of oil by high pressure CO2 [3] and comprehensive reviews
of oil solubility in CO2, seed oil extraction using supercritical fluids and
fractionation of oils have been published in the 1980s and 90 s [4–6]
and more recently in 2012 [7]. Despite this early R&D work, extraction
of commodity oils at a large scale using this technology was found to be
infeasible because of the cost, batch-wise extraction, non-availability of
very high pressure plant and competing solvent extraction technology.
One notable exception is the extraction of roasted sesame seed oil in
Korea, which is carried out in large scale supercritical CO2 extraction
plant by U-max and Ottogi [8,9]. Roasting has no effect on the ex-
traction of the oil, and is beneficial for co-extraction of the anti-oxidant
sesamol, which is improved by using a 1% water co-solvent [8]. Su-
percritical extraction of seed oils has generally been limited to high
value speciality oils for nutraceutical, cosmeceutical, dietary supple-
ment and animal health products. The competing extraction technology
is cold-pressing. The high value oils typically contain high levels of
polyunsaturated fatty acids and/or seeds that are not amenable to cold-
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The Journal of Supercritical Fluids 134 (2018) 260–268
Fig. 1. Schematic of current extraction supercritical
solvent and potential future solvents for processing
of high value lipids.
pressing. Typically, the seed and berry oils are extracted in small bat-
ches in multipurpose extraction plants. The extraction of oil is generally
solubility limited and so the extraction pressure used is close to the
maximum that can be achieved in the extraction plant. Oils that have
been extracted in New Zealand and elsewhere include those rich in
alpha-linolenic acid (ALA) or gamma-linolenic acid (GLA) such as
evening primrose oil, borage seed oil, blackcurrant seed oil, kiwifruit
seed oil, perilla seed oil, sea buckthorn oil; oils that contain unusual
polyunsaturated fatty acids, such as Biota Orientalis oil, sandalwood
seed oil, pomegranate seed oil; and oils that contain other active in-
gredients such as rosehip seed oil. We have found that roasting and
cooking processes for seeds can make the oil more difficult to extract
using cold-pressing, but has no effect on extractability using super-
critical CO2 extraction. The benefits of CO2 extraction are that there is
less extraction of chlorophyll, the omega-3 fatty acids are extracted in
an oxygen free and high heat-free environment and there is no ex-
traction of phospholipids compared with cold pressed or hexane-ex-
tracted oils. Supercritical CO2 extraction is a good extraction tech-
nology when seed oil content is low, the seed is too hard or the seed
moisture content is too low for cold pressing, and/or extraction of seed
with both seed oil and volatile essential oil is being carried out as two-
stage pressure reduction can be used to give a seed oil and a volatile
essential oil [10,11].
The issues with supercritical CO2 extraction are mainly to do with
cost and throughput. Under standard extraction conditions of around
300–500 bar and 313–333 K the seed oil solubility is around 8–20 g/kg
of CO2 [7], and so a high solvent usage is required to extract the oil,
which limits throughput if the seed has a high oil content. Water is also
co-extracted, and this causes issues in both removal from the final
product and degradation of the oil. There can be some generation of
free fatty acids (FFA) due to the co-extraction of water, typically in the
O. Catchpole et al.
range 1–5% of total oil extracted. We postulate that there are two
possible mechanisms for the generation of FFA: the presence of natural
lipases in the ground seeds that are exposed to the substrates and sol-
vent due to prior grinding of the seed that result in enzyme catalysed
production of FFA in a manner analogous to the use of commercial
immobilized lipases in supercritical CO2 to produce FFA [12]; and the
acid-catalysed generation of FFA due to CO2 dissolving into the pre-
cipitated aqueous/oil dispersion at separator pressure conditions. The
use of two-stage separation (2 separators in a pressure cascade) can
drive most of the water and some of the free fatty acids into second low
pressure separator and most of the oil into the first high pressure se-
parator, but the partitioning is incomplete. Water that is co-extracted
and co-precipitated with oil partly separates out from the oil in the first
separator vessel, and can be incompletely recovered by careful opera-
tion of the separator outlet valves as it is more dense than the oil and
comes out first. The remainder of the water can be removed by cen-
trifugation or more preferably by vacuum evaporation at moderate
temperature to give a clear oil. Other processing problems include by-
passing, bed compaction and/or generation of fines which can lead to
blocking of filters and basket deformation, which are more likely to
occur with high oil content seeds. Polyunsaturated oils extracted by
supercritical CO2 or by cold-pressing are in general highly unstable.
This necessitates immediate extraction of seeds after grinding; and
addition of antioxidants to the final product unless the extracted oil is
also rich in natural antioxidants. The recovered oils then need to be
stored under an inert gas blanket in lined drums.
2.2. Marine oils and omega-3 concentrates
The extraction of marine oils and concentration of omega-3 fatty
acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
from mostly marine sourced feed materials using supercritical fluids is
another area of extensive R&D investigation. Despite considerable
promise, this is an area in which there has been little commercial de-
velopment aside from KD-Pharma and Nutrizeal/Pharmalink Extracts
Limited. A substantial amount of work has been performed on super-
critical technologies to concentrate polyunsaturated fatty acids (PUFA)
from fish and more recently algae oils. The main fractionation tech-
nologies have been reviewed previously [1,13], aside from recent de-
velopments in supercritical chromatography. Therefore, the extraction
of green shell mussel oil, and the concentration of PUFA using super-
critical chromatography are reviewed below. The commercial extrac-
tion of NZ Greenshell mussel neutral lipids has been carried out for a
long period of time in New Zealand by Nutrizeal (now Pharmalink
Extracts Ltd), and there is a further product made by Aroma (New
Zealand) now that patents have expired [14]. In the commercial pro-
cess, freshly harvested and rinsed green shell mussels are shucked using
a high pressure process, the flesh is recovered, coarsely minced and
stabilized with tartaric acid, frozen and then freeze-dried before being
extracted with supercritical CO2. The resultant mussel extract (com-
mercial name PCSO-524 meaning Perna Canaliculus Supercritical Oil
containing 524 different fatty acids) is blended with olive oil at a 1:2
ratio with some added Vitamin E to give the final commercial product
Lyprinol™. The anti-inflammatory activity of the extracts [14–22] has
been variously attributed to novel C20:4 fatty acids [18], free fatty
acids [19,20], N-acyl ethanolamines [21], and furan fatty acids [22].
The mussel oil yield is around 5% of the freeze-dried mussel powder.
The extraction process is slow due to the low solubility of the extract
components in supercritical CO2. The oil consists of a mixture of tri-
glycerides and partial glycerides, free fatty acids, sterols, sterol esters
and carotenoids [20,23]. The extract triglyceride and free fatty acid
fractions have high levels of omega-3 fatty acids, typically 20% EPA
and 12–15% DHA [23,24]. The overall fatty acid composition is ex-
tremely complex [24]. Greenshell mussels also contain phospholipids at
a level similar to the neutral lipids. The composition of these phos-
pholipids is very complex [25]. They can be extracted using dimethyl
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ether (DME) [26].
In general, an oil or lipid mixture that contains PUFA to be con-
centrated by supercritical fluid chromatography must be first converted
to ethyl esters or free fatty acids; unwanted reaction products and
contaminants are removed, and then one or more fractionation pro-
cesses are employed to concentrate the PUFA and reduce the saturates
and mono-unsaturates. The nutraceutical market has moved to con-
centrates containing ≥60% EPA + DHA with various ratios of EPA to
DHA depending upon the starting material and health outcome (high
EPA for brain health, high DHA for joint and heart health); and the
pharmaceutical market to ≥95% EPA or DHA. Advances in super-
critical fluid chromatography stationary phases have now made it
possible to perform single step chromatographic fractionation to
achieve very high concentration levels [27,25]. We have recently ap-
plied this technology to unusual fatty acids including non-methylene-
interrupted PUFA such as juniperonic acid (JA); and ximenynic acid
(XA), which contains a triple bond [29]. The practical and economic
issues with production scale supercritical chromatography relate to the
cost of the specialized stationary phases, decreased separation effi-
ciency during scale-up and throughput. The separation efficiency is
highly dependent upon particle size (the smaller the particle the better),
but so is the pressure drop across the column. Special coated packings
specific for use with supercritical CO2 are only supplied in 5–10 μm
particles sizes, and are currently prohibitively expensive at a produc-
tion scale. Thus, uncoated silica becomes the default packing, resulting
in decreased performance [27–29].
2.3. Carotenoid-rich extracts
Carotenoids are another class of lipids that are currently highly
valued by industry because of their human health benefits. These health
benefits span a broad range of activity, from lipophilic antioxidants
such as astaxanthin through to eye health such as lutein [30,31]. Car-
otenoids are further classified as carotenes, which are purely hydro-
carbons (e.g. lycopene, β-carotene) and xanthophylls, which contain
oxygen in the form of hydroxyl, carbonyl and epoxide groups, two beta-
ionone groups that are connected by a highly unsaturated hydrocarbon
chain (e.g. astaxanthin, zeaxanthin). All carotenoids have very low
solubility in supercritical CO2 under standard operating pressures of
300–500 bar. The extraction of carotenoids is thus favoured by high
pressures and/or use of co-solvent, or the use of alternative near-critical
extraction solvents such as propane or dimethyl ether (DME). The main
carotenoids found in commercial products are β-carotene, lycopene,
lutein, canthaxanthin, zeaxanthin and astaxanthin. The main in-
dustrially-utilized natural sources of carotenoids are flowers, micro and
macro algae, some marine biomasses, and certain microorganisms [32].
The main carotenoid-rich extract produced by supercritical CO2 ex-
traction is astaxanthin-rich oleoresin from the microalgae Haemato-
coccus pluvialis. Astaxanthin is present in the oleoresin mainly in the
form of mono esters, and can reach up to 10% or more (free astaxanthin
basis) of the oleoresin. Astaxanthin mainly in the form of di-esters is
also present in krill oil, which is discussed separately. Astaxanthin
oleoresin is or has been produced commercially at NateCO2 (part of
Hopfenveredlung, Germany) and Valensa (US) in bespoke ultra-high
pressure CO2 extraction plant, Pharmalink Extracts Limited (New
Zealand) and Fujifilm (Japan). The combination of lutein and zeax-
anthin is often targeted at the treatment and prevention of age-related
macular degeneration [33]. In nature, these compounds are most
abundant in the flower petals of yellow Marigold flowers (Tagetes erecta
L.), in which they are chemically bound to fatty acids including lauric
and palmitic acids [34], making Marigold a good candidate for com-
mercial industrial extraction using supercritical CO2. Fit Ingredients
e.K. (based in Germany) market a lutein ester and zeaxanthin ester
product obtained from marigold flowers by supercritical CO2 extraction
in India by Spisyslimited. Extraction pressures of ≥1000 bar are gen-
erally considered “ultra-high pressures”, with the main advantage being
O. Catchpole et al.
the enhanced solubility of poorly soluble compounds such as car-
otenoids that would otherwise require the use of a co-solvent [35–37].
Valensa, a manufacturer of dietary supplements based in Florida USA,
utilizes ultrahigh pressure CO2 for extraction of carotenoids such as
astaxanthin, lutein and zeaxanthin, which are then used in a range of
products aimed at joint, eye, cardiovascular and brain health. Ultrahigh
pressure CO2 extraction is also commercially used by NateCO2 to ex-
tract carotenoids from algae, marigold or paprika.
An alternative to ultrahigh pressure extraction is the use of CO2 with
a co-solvent such as ethanol, which will increase the polarity of the
solvent and favour the extraction of more polar compounds at moderate
pressures [38,39]. Vegetable oils, particularly triglycerides, can also act
as co-solvents [40]. However, these options introduce a new material in
the extract, which may need to be removed to meet product specifi-
cations. A further option is the use of alternative solvents such as
subcritical dimethyl ether (DME) or propane, which provide higher
solubility for carotenoids. The use of such solvents has become rela-
tively common in recent years [41–43], but commercial applications
still need to be further developed. The solubility of β-carotene in pro-
pane has been reported to be approximately 2 orders of magnitude
higher than that in supercritical CO2 [44]. In another study, DME
provided virtually complete extraction of carotenoids (mainly can-
thaxanthin) from chilli powder [45]. In the particular case of H. plu-
vialis, we have observed that the extraction yield of oleoresin containing
astaxanthin obtained with DME or propane does not vary significantly
from CO2 at 500 bar but the extraction rate is greatly enhanced as
shown in Fig. 2. The extraction of H. pluvialis with CO2 to obtain oil
with no or low astaxanthin followed by DME to give a concentrated
astaxanthin oleoresin, or extraction of the total oleoresin with DME
followed by re-extraction of the oleoresin with CO2 to obtain and oil
extract with no or low astaxanthin and a residue with high astaxanthin
content has also been demonstrated [46].
The main competition to natural carotenoid extracts comes from
chemically synthesized carotenoids. Astaxanthin and lutein can be in-
dustrially produced by chemical synthesis at a lower price than
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naturally sourced carotenoids. There is considerable debate about the
merits and biological activity of synthesized versus natural carotenoids
as the products are not exactly identical. Synthesized astaxanthin
contains some isomers that are not generally found in nature. Currently,
human health products are predominantly based on naturally-sourced
carotenoids based on the perception that ‘natural is best’. However,
addition of synthesized carotenoids to animal feed diets such as farmed
salmon (to give pink flesh) and chickens (to give a yellow to orange
colour in egg yolks) is routinely performed because of the cost ad-
vantage and thus the synthesized carotenoids enter the human diet via
consumption of salmon or eggs respectively.
2.4. Complex lipid-rich extracts
Phospholipids and glycolipids are an essential component of cellular
membranes in animals and plants respectively. Phospholipids in general
consist of a glycerol backbone, two fatty acids esterified to glycerol at
positions 1 and 2, and a phosphate group at position 3. The phosphate
group is further esterified to an amino alcohol (choline, ethanolamine,
serine) or a polyol (inositol or glycerol). Glycolipids in general consist
of one or more carbohydrate groups linked to a sphingosine or glycerol
backbone. Complex lipids typically form part of cell membranes and
can interact strongly with both proteins and polysaccharides, making
them difficult to extract [47]. Polar solvents are required to sufficiently
overcome hydrogen and ionic bonding forces. In some cases complex
lipids may also be physically trapped within a tissue matrix. Water is
effective as a co-solvent in this case, however is not suitable for use with
CO2. In other cases substantial destruction or denaturation of the sub-
strate (for example through homogenisation or the action of a protease)
is effective. [26,48–50]. The main high value phospholipid-rich product
that has been commercially produced using supercritical CO2 is krill oil.
Dairy, egg yolk and marine biomasses are other sources of phospholi-
pids of research and commercial interest [26,51–54]. The use of CO2 to
fractionate and concentrate solvent-extracted glycolipids (mono- and
di-galactodiglycerides) from neutral lipids has also been reported [55].
Fig. 2. Extraction curves for freeze-dried Haematococcus pluvialis using
CO2 (500 bar), DME (40 bar) and propane (40 bar), flakes are flakes of
freeze-dried algae as received from the freeze-drier, powder is the ground
flakes [unpublished data].
O. Catchpole et al.
A co-solvent, usually ethanol, is required to extract phospholipids using
supercritical CO2. Krill oil, which is commercially extracted by Phar-
malink Extracts Limited, is herein discussed to describe the general
principles. Krill oil is in general around 40% phospholipids rich in the
omega-3 fatty acids EPA and DHA, triglycerides which are relatively
low in EPA and DHA, and up to 1 g/kg astaxanthin. Krill is harvested
mainly in the Antarctic, and either immediately processed to a meal on-
board special factory ships or deep frozen. When processed im-
mediately, the krill is deshelled, cooked, pressed to obtain a neutral oil,
treated with a protease, centrifuged to separate out non-lipid material
and then drum dried to give pellets which are highly enriched in total
lipids (40–45% dry basis) of which about half are phospholipids. Cu-
mulative extract yield curves for the pilot scale extraction of krill oil
from krill meal using a two-step, two separator supercritical CO2 pro-
cess and a single step DME process [26] are shown in Fig. 3. In the first
step of the pilot scale supercritical CO2 process, krill meal is extracted
with CO2 + 5% ethanol (300 bar, 333 K) or CO2 alone (500 bar) to
extract mostly neutral lipids. In the second step, the ethanol co-solvent
concentration is then increased to greater than 10% to extract the
phospholipids [56]. The bulk of the krill lipid extract in each step of the
process is collected in the first (S1) separator, and the co-solvent in the
second (S2) separator. A portion of phospholipid is carried over into the
second separator, and there is a considerable cost incurred in evapor-
ating the co-solvent to recover it. Also included for comparison in Fig. 3
is a supercritical CO2 extraction in which the co-solvent from a previous
run recovered from the S2 separator during the second step was re-
cycled and used as the co-solvent without recovering the dissolved
extract, to see if the recycled S2 extract could be recovered in the first
(S1) separator along with the new extract. This was achieved, as shown
in Fig. 3. The apparent yield in S1 has increased, but this is due to the
recovery of the recycled extract and there is no change in the actual
yield from the fresh krill meal. The S2 co-solvent with dissolved extract
can only be recycled a few times, as some water is also co-extracted
from the krill biomass which then accumulates in the recycled co-sol-
vent. The water in the ethanol decreases its effectiveness as a co-
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The Journal of Supercritical Fluids 134 (2018) 260–268
Fig. 3. Extraction curves for krill meal using a two step extraction
and 2 separator process using CO2 at 333 K, 300 bar and 5%
ethanol co-solvent (first stage) and 20% ethanol co-solvent
(second stage); two stage extraction and 2 separator process using
CO2 at 313 K, 500 bar and no co-solvent (first stage) and 16%
ethanol co-solvent (second stage); and single stage, single se-
parator DME extraction at 40 bar, 313 K. Legend: F = fresh
ethanol co-solvent, R = recycled S2 ethanol co-solvent from a
previous run containing dissolved extract, S1 = first separator,
S2 = second separator [unpublished data].
solvent. The yield of total extract using DME is higher, and the ex-
traction time and DME usage substantially lower than that with
CO2 + ethanol co-solvent. However, DME is flammable, and DME sol-
vent residues in the extracts must be reduced by vacuum evaporation to
meet regulatory requirements, and in the extracted solids to meet safe
disposal requirements.
One of the advantages of extracting phospholipids with CO2 and an
ethanol co-solvent is that the residual defatted biomass (enriched in
protein) is generally still suitable for use as a secondary product, which
may not be the case for alternative liquid solvent extracted substrates.
Difficulties associated with CO2 and co-solvent extraction include
higher downstream processing costs to evaporate and recover ethanol,
greater solvent consumption, and the need to accommodate flammable
solvent handling in the process design and operation. CO2 solvent losses
are increased because CO2 dissolves at high levels in the ethanol in the
extract phase and cannot be completely recovered in a separation
process. Ethanol losses can also be high due to retention of ethanol in
the residual biomass, and the progressive concentration of con-
taminants (primarily water) in the ethanol limiting the recyclability of
the ethanol. As noted in our previous works [1,56], the solubility of a
phospholipid in supercritical CO2 and ethanol co-solvent depends on
the type of polar group. The most soluble phospholipids are phospha-
tidylcholine (PC) and sphingomyelin (SM) and their analogues alkyla-
cylphosphatidylcholine (AAPC) and dihydrosphingomyelin (DHSM),
followed by phosphatidylethanolamine (PE). The phospholipids phos-
phatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL) have
very low to negligible solubility. Partially hydrolysed phospholipids in
which the fatty acid at position 1 or 2 has been hydrolysed (known as
lyso-phospholipids e.g. LPC), have much lower solubilities than the
parent phospholipid. In contrast, DME is a non-selective solvent for
phospholipids [26,52] and all phospholipids are extracted in proportion
to their concentration in the feed material. DME is also able to extract
phospholipids from aqueous streams and wet biomasses and thus offers
two significant processing advantages over supercritical CO2 + co-sol-
vent. The co-extracted water when extracting wet biomass or an
O. Catchpole et al.
aqueous stream with DME acts as a co-solvent that reduces the solu-
bility of triglycerides, offering some selectivity in extraction. As noted
earlier, there are also issues with the use of DME including flamm-
ability, need to meet regulatory residue level requirements in products
from the extraction process, and lack of industrial scale extraction fa-
cilities that can use this solvent. We have recently constructed a de-
monstration scale facility to overcome some of these issues.
3. Issues regarding future developments and gaps in knowledge
Most of the issues facing expansion of the supercritical extraction
industry in regard to high value lipid products and other natural pro-
duct-type extracts are non-technical in nature. Some of these con-
straints include regulatory-type issues, Intellectual Property/Freedom
to Operate (IP/FTO) constraints, and competing technologies.
Technical barriers include a lack of scale-up data, inability to produce
food-grade samples, lack of partnerships between academia or research
institutes and industry. The regulatory constraints are not unique to
supercritical extraction. Extracts that are to be used for human con-
sumption must be made in supercritical extraction facilities that have
the required regulatory certification to meet the requirements of the
market that they are to be sold in. However, there are separate reg-
ulations for food use i.e. when the extract (e.g. hop extract for fla-
vouring beer) or residual extracted material (decaffeinated coffee
beans) are deemed to be for use in foods or beverages; or when the
extract is a dietary supplement, nutraceutical or functional food in-
gredient. Food regulations are stricter than for dietary supplements,
because the food can be eaten in far greater amounts and thus the ex-
posure to potentially harmful substances is much greater. As an ex-
ample, omega-3 extracts can be used to fortify foods and so production
must meet the relevant food regulations, or they can be an over-the
counter (OTC) dietary supplement product in the form of gelatine
capsules, or they can be a pharmaceutical product and production must
meet pharmaceutical GMP regulations. Each form of regulation has its
own compliance costs and time frame to achieve compliance for a new
product. The supercritical extraction solvent must also be approved for
food use or pharmaceutical use, but there are often no useful guidelines
to establish compliance for dietary supplement use.
The solvents approved for food use are generally specific to a
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Fig. 4. Solubility of seed oil in CO2 at high pressures and 3 temperatures.
Filled data points – this work on apple seed [57]; hollow data points lit-
erature data on soya oil [2].
country or region [57]. DME has relatively recently been approved for
general food use in Australia and NZ [58] and for limited food use in
Europe [59], and is currently undergoing a GRAS evaluation in the US
[60]. Propane is a permitted extraction solvent in many countries, but is
approved only for aerosol food use in the US. The solvents which are
generally regarded as being safe under GMP for production of phar-
maceuticals are given by the International Council for Harmonisation
(ICH) regulations [61]. No new ‘safe’ solvents have been approved for
some time, and the list of ICH safe solvents does not include any near-
critical solvents such as supercritical CO2, even though it is used for
production of some pharmaceuticals. A further regulatory barrier is the
requirement to also get the extract itself approved for human con-
sumption if it is to be incorporated into a food product. The company
selling the extract or food ingredient must generate a dossier demon-
strating that the product is safe to consume which includes animal
toxicity studies performed using the product and/or relevant corro-
borating literature data, method of production, quality control, detailed
composition including contaminants; or equivalence to an existing
product which has already been approved. A further significant cost can
be incurred in establishing health claims for a new product, as clinical
trials are required. The development and launch of new products is also
hindered by excessive patenting. Many patents and patent applications
are filed as blocking patents that can have the aim of preventing a type
of processing technology being used for production. A notable example
is the production of krill oil using supercritical technology [62–64].
4. Future perspectives
4.1. Seed oils
Research and development work is being performed using ultra-
high pressure CO2 extraction [65], and use of alternative extraction
solvents such as propane [66–69] or DME to extract seed oils. The ex-
traction rate for seed oils is greatly accelerated using high pressures
(> 500 bar) and moderate to high temperatures. Solubility isotherms
for triglyceride and wax ester oils were first reported by Quirin [2] at
pressures up to 2000 bar. This early work showed that oils become
miscible with CO2 at pressures in the range 1000–2000 bar and tem-
peratures of about 353–373 K. Our own work, at pressures of up to
O. Catchpole et al.
1300 bar and temperatures in the range 318–338 K, also show a dra-
matic increase in solubility for apple seed oil at high pressure and the
highest temperature, as shown in Fig. 4.
The amount of water co-extracted decreases markedly as the ex-
traction pressure is increased. The solubility of water does not increase
markedly with pressure, and so the amount of water extracted is re-
duced because a substantially lower amount of CO2 is required to ex-
tract all the oil. The very large difference in solubility with temperature
and high pressure makes an isobaric extraction and separation process
possible, with extraction taking place at high temperature, and se-
paration at low temperature. Alternatively, an isothermal process in-
volving extraction at high temperature and high pressure of
≥1000 bar, and separation at moderate pressure of 250–300 bar.
We expect to see the commercial use of high pressure extraction or
alternative extraction solvents to produce high value seed oils in a more
cost-effective manner. As with marine oils, we can expect to see that the
base oils become a commodity, and that concentrates of particular
bioactive fatty acids such as alpha-linolenic acid, gamma-linolenic acid,
juniperonic acid (JA), sciadonic acid, ximenynic acid (XA) become the
high value products. Concentrates of some fatty acids are already
commercially available via Fit ingredients e.K. This favours integration
of processing steps such as extraction followed by in-situ conversion to
free fatty acids or ethyl esters and possibly chromatographic separation.
A new opportunity may arise to process oils from genetically modified
seeds to enable plant-based manufacture of omega-3 rich oils, although
this will be prevented by the regulatory environment in many countries.
Science and technology is advancing in the production of seed oils
containing EPA and/or DHA [70–72]. Supercritical chromatography is
a very promising technology to obtain very highly concentrated fatty
acids or single fatty acids. There is considerable scope for investigating
new stationary phases that have specific interactions with double and
triple bonds of polyunsaturated fatty acids. Our own work has de-
monstrated that production of very highly pure ethyl esters of EPA,
DHA, XA and JA using pilot scale supercritical chromatography
[27–29] using solid phases with special surface coatings designed for
use with supercritical CO2.
4.2. Marine oils and omega-3 concentrates
We expect to see the processing of oils obtained from micro and
macro algae to be a growing area. There is substantial investment into
the industrial scale heterotrophic and photobioreactor production of
microalgae to produce oils that are highly enriched in a single poly-
unsaturated fatty acid. DHA-rich oils have been produced for a con-
siderable amount of time by Martek (now owned by DSM) and the
extract incorporated into infant formula. Algae that produce EPA are
the next commercial target. The processing challenges are the high
water content and the resistance to mass transfer that is generated when
the microalgae is dried, high costs to produce biomass, and the form of
the lipids. The PUFA are usually present in the complex lipid fraction of
the lipids (mostly as mono-and digalactosyldiglycerides) and are thus
not extractable with CO2 and poorly extractable with CO2 + cosolvent
but can easily be extracted with DME [73]. The lipids then require
conversion to ethyl esters, purification to remove non-lipid material,
reaction products and colour bodies. Extensive research is now being
performed into the use of DME as an extraction solvent for wet algal
biomass [43,73–75], taking advantage of the partial miscibility of DME
+ water. We have demonstrated the extraction of EPA-rich oils and
production of 95% EPA using DME as the extraction solvent from
wet algae [27,73]. A recent work has shown that it is possible to per-
form methanolysis of triolein in the solvent mixture DME + methanol,
using KOH as the catalyst [76]. This opens the opportunity for the si-
multaneous extraction and conversion of lipids to ethyl esters from dry
algae using DME/ethanol/KOH; or the simultaneous extraction and
conversion of lipids to free fatty acids from wet algae using DME/
ethanol/KOH.
266
The Journal of Supercritical Fluids 134 (2018) 260–268
4.3. Carotenoid-rich extracts
Due to their versatile health-promoting properties, the global
market demand for carotenoids is estimated to increase from US$1.5
billion in 2014 to US$1.8 billion in 2019 [30]. We expect to see an
increase in the types of carotenoid-rich extracts that are produced, and
in particular, fucoxanthin-rich products. Fucoxanthin can be extracted
from brown seaweed [77], but production by microalgae seems a more
promising route to higher yields and lower costs. Current market trends
indicate increased demand for natural-based products, as chemically
synthesized carotenoids have lower acceptability to health-conscious
consumers. Microbial production of carotenoids is expected to grow in
the future, although in spite of the intense research, only three have
reached commercial scale so far: β-carotene by the alga Dunaliella sp,
astaxanthin by the alga Haematococcus pluvialis, and β-carotene by the
fungus Blakeslea trispora [32]. Other natural sources such as Gac fruit
(Momordica cochinchinensis Spreng.), one of the richest natural sources
of lycopene, lutein and β-carotene [78], and different agro-industrial
waste (e.g. tomato, mango, carrot, pink guava, banana) [30] are also
expected to be exploited in the future. Currently, the number of com-
panies entering the astaxanthin-rich oleoresin market has been growing
rapidly, and it is likely that the market is becoming mature. New pro-
ducts will emerge as the fermentation and harvesting technology is
applied to other algal biomasses that produce different carotenoids, and
also through biotechnological processes using yeasts, filamentous fungi
or bacteria. However, there need to be some breakthroughs in reducing
the cost of producing biomass by phototrophic fermentation (use of
light as the energy source and CO2 as the carbon source). There is likely
to be increasing competition from synthesized astaxanthin, and possibly
organisms that have been genetically modified to over-produce the
desired carotenoid. A completely synthesized astaxanthin product for
human use is being produced and marketed by ZanthoSyn (https://
zanthosyn.com/) but the product itself does not seem to be backed by
any published studies. The application of GM-technologies to improve
the photosynthetic process in micro-algae could greatly improve bio-
mass production times.
4.4. Phospholipid-rich extracts
There is a growing interest in phospholipids for human health as a
source of both omega-3 fatty acids and also of the polar alcohol e.g.
choline. There is also a growing interest in the use of phospholipids as
formulating agents to improve the bioavailability of lipophilic bioac-
tives. While supercritical CO2 + co-solvent has some significant lim-
itations in terms of the cost and complexity of the extraction process,
we see tremendous potential for the use of DME or propane to extract
phospholipids. The extraction of, for example, krill lipids can be per-
formed in a single step, yielding an extract rich in phospholipids and
astaxanthin and with relatively low levels of trimethylamine oxide
(TMAO). Similarly, it is a simple process to obtain dairy phospholipids
rich in phosphatidylserine (PS) and sphingomyelin (SM) by direct ex-
traction of dry beta serum powder, particularly if the lactose content
has already been reduced by diafiltration [52]. Similarly, egg yolk
phospholipids are easily extracted from dry egg yolk powder. We have
also observed that DME seems to act in a manner similar to water when
separating neutral lipids from hydratable phospholipids. When egg yolk
lipids are extracted and then the total extract recovered by pressure
reduction, phase separation into a neutral lipid light phase and phos-
pholipid rich heavy phase occurs [26], and the top oil phase can be
easily decanted or separated by centrifugation. DME appears to hydrate
the phospholipids. The DME is easily removed by heating or vacuum.
Another major benefit of DME is that wet biomass can be extracted, this
avoiding the drying step. Phospholipids and glycolipids are more so-
luble in the combined solvent mixture DME + water than neutral lipids
and are preferentially extracted from an aqueous stream. This makes
possible the processing of a feed stream containing a complex lipid
O. Catchpole et al.
source such as a dairy stream, fermentation biomass, microalgae, egg
yolk [26,50,51,73]; and integrated processing in which the lipid-
bearing feed stream has been chemically and/or enzymatically treated
to make a pumpable slurry [50,77]. Typically, this may be a protease to
partially hydrolyze proteins into peptides, lipases to break up neutral
lipids, and carbohydrases to hydrolyze complex carbohydrates. Further
upstream processes can be performed, to separate small fragments from
larger compounds e.g. peptides from proteins by membrane fractiona-
tion whereby the lipids are recovered in the retentate [53]
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