Eur. J. Lipid Sci. Technol.

2009, 111, 135–141 135

Research Paper
Comparison of low-temperature processes for oil and
coenzyme Q10 extraction from mackerel and herring

Serge Laplante1, Nathalie Souchet1 and Piotr Bryl2

1
Département de Biologie, Chimie et Géographie, Université du Québec à Rimouski, Rimouski, Canada
2
Centre Technologique des Produits Aquatiques, Ministère de l’Agriculture des Pêcheries et de l’Alimentation
du Québec, Gaspé, Canada

Among the fat fish species available from Eastern Quebec (Canada), whole Atlantic mackerel (Scomber
scombrus) and herring (Clupea harengus) represent abundant fishery resources which are currently under-
utilized. They have relatively high contents of oil and coenzyme Q10 (CoQ10) in their tissues, which
could be valuable for nutraceutical applications. Therefore, two low-temperature extraction processes
were compared for the recovery of oil and CoQ10 from these resources, such as enzymatic hydrolysis
using Protamex and supercritical CO2 (SCO2) using fish lyophilizates. The results revealed that highest
yields of oil and CoQ10 were obtained using the enzymatic hydrolysis process with mackerel. Whatever
the process used, CoQ10 concentrations were higher in herring oil, due mainly to a more selective
extraction of CoQ10 over that of the oil. The highest CoQ10 recovery rates (extraction efficiencies) were
obtained using the enzymatic hydrolysis process with both types of fish, but also the SCO2 process with
herring under some conditions. For mackerel, the lower CoQ10 recovery rates obtained from the SCO2
process were explained by its more important matrix effect. An economic assessment of both processes
revealed that the enzymatic hydrolysis extraction process would be the most promising for up-scaling the
recovery of oil and CoQ10 from these resources.

Keywords: Coenzyme Q10 / Enzymatic hydrolysis / Extraction / Fish / Supercritical CO2

Received: May 23, 2008; accepted: September 27, 2008
DOI 10.1002/ejlt.200800133

1 Introduction of the oral administration of CoQ10 were reported, in partic-

In Eastern Quebec, only 60% of the available landings of
whole mackerel and herring (,6000 t/year) are actually pro-
cessed industrially [1, 2]. The rest of the catch is mainly used
as a lure for lobster and crab pots, which represents a limited
valorization [2]. Therefore, the valorization of these resources
could emerge from the development of efficient extraction
processes for the recovery of value-added products, such as
polyunsaturated fatty acid-rich oils [3–9]. Moreover, high
contents of another health-promoting lipid, the coenzyme
Q10 (CoQ10), were found in muscle tissues of these fish
species [10–13]. Numerous beneficial and therapeutic effects
Correspondence: Serge Laplante, Centre Technologique des Produits
Aquatiques, 96 Montée de Sandy Beach, bureau 1.07, Gaspé, Québec
G4X 2V6, Canada.
E-mail: serge.laplante@partenaires.mapaq.gouv.qc.ca
Fax: 11 418 3608514
Phone: 11 418 3687655
ular the protection from diseases related to the general aging
process such as cardiovascular diseases, diabetes and cancer
[14–17].
Due to the susceptibility of those lipids to oxidation, low-
temperature extraction processes, such as supercritical carbon
dioxide (SCO2) and enzymatic hydrolysis are envisaged. The
SCO2 process is regarded for extraction and fractionation of
fish oils due to the fact that CO2 is inexpensive, clean, non-
toxic, non-flammable, and has a low critical temperature of
31 7C [18–21]. Enzymatic hydrolysis for fish oil extraction
using food-grade proteases could provide an inexpensive
process that favors the release of lipids from protein matrix
[22]. It does not compromise the nutritive value of lipids, and
it is widely used in the food industry [23]. Many studies have
been carried out on fish oil extraction under this type of pro-
cess [22–32].
To our knowledge, no data are available on the CoQ10
content in fish oils extracted from both processes. Moreover,
the presence of lipid-like antioxidants such as CoQ10 in fish

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136 S. Laplante et al.
oil extracts is important to consider, not only for heath bene-
fits but also for protection of polyunsaturated fatty acids
against oxidation. Therefore, the objective of this study was to
compare the performances and production costs of two low-
temperature extraction processes (SCO2 vs. enzymatic
hydrolysis) for the recovery of oils and CoQ10 from mackerel
and herring. This will provide information about which pro-
cess to choose in view of an extraction on a larger scale.
2 Materials and methods
2.1 Sources of fish
Whole Atlantic mackerel (Scomber scombrus) and herring
(Clupea harengus) were the fishery resources (fish) used in this
study. They were caught in September 2005 and 2006 from
Baie-des-Chaleurs (Qc, Canada) and were obtained from
Lelièvre, Lelièvre & Lemoignan Ltée., Ste-Thérèse-de-Gaspé
(Qc, Canada). They were put on ice, immediately shipped to
the laboratory and kept frozen at –40 7C under vacuum
packing before use. They were then ground for the enzymatic
hydrolysis process, and a part of the grinding was lyophilized
for the SCO2 extraction process. The global composition and
CoQ10 content of the fish and their lyophilizates are summa-
rized in Table 1.
2.2 Global composition of fish and lyophilizates
Total lipid content was determined using methanol/chloro-
form extraction [33]. Protein content was determined by the
Kjeldahl method (% nitrogen66.25) [34]. Humidity was
determined after drying overnight at 105 7C, and ash content
was determined after heating the samples overnight at 550 7C
[34].
2.3 Determinations of CoQ10 in samples
Determinations of CoQ10 in samples were done using an
HPLC procedure with photodiode array detection, as report-
Table 1. Composition of fish and lyophilizates used for the CoQ10 extraction processes.
Type of sample Composition of samples
Humidity Lipid
[wt-%] [wt-%]
Mackerel 64.58 6 0.04 16.00 6 0.25
Herring 71.41 6 0.32 10.41 6 0.60
Mackerel lyophilizate 1.49 6 0.05 42.60 6 0.30
Herring lyophilizate 1.48 6 0.07 39.31 6 0.16
Data are means 6 standard deviation from duplicates (n = 2).
 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2009, 111, 135–141
ed earlier [13]. The described procedure used homogeniza-
tion with sodium dodecyl sulfate and NaCl solutions followed
by ethanol/hexane extraction for solid and aqueous samples,
whereas oil samples were directly dissolved in isopropanol
solvent prior to HPLC analysis.
2.4 Extraction by enzymatic hydrolysis process
For each experiment, 100 kg of fish was ground up in two
steps. In the first step, a crude grinding was done using a
Comitrol 3600 grinder (cutting head model 3-025040-U;
Urschel Laboratories Inc., Valparaiso, IN, USA), followed
by a finer grinding step with another cutting head (model 3-
K-030120; Urschel Laboratories Inc.), giving particles of
approximately 1 mm in diameter. The enzymatic hydrolysis
process used Protamex (Novozymes Ltd., Bagsvaerd,
Denmark) as the commercial source of protease, according
to the following conditions (pH = 7.0, temperature = 45 7C,
2.5 h hydrolysis, 1 g enzyme/kg fish, 1 kg water/kg fish),
with sufficient agitation to keep mixture homogeneity. This
was followed by a heat treatment step in a plate heat
exchanger system for inactivation of enzymes (90 7C,
1 min). Most of the solids from the hydrolyzate were elimi-
nated using a clarifying decanter (model CA 220-01-33;
Westfalia Separator Inc., NJ, USA), and the oil fraction was
recovered by continuous centrifugation at 11,0006g (model
LAPX 404; Alfa Laval, Lund, Sweden). In order to remove
most of the residual aqueous phase (1–2 wt-% of total
composition), it was then clarified using batch centrifuga-
tion at 10,0006g during 15 min (model J2-HC; Beckman,
CA, USA). The oil was weighed and kept frozen under
nitrogen atmosphere at –80 7C before analysis. Each treat-
ment was repeated twice.
2.5 Supercritical extraction system
The SCO2 extractions were done at Centre d’Études des Pro-
cédés Chimiques du Québec (CEPROCQ), Montréal, Qc,
Canada. The extraction reactor was a high-pressure SS 304
cylindrical vessel having an internal volume of approximately
Protein Ash Coenzyme Q10
[wt-%] [wt-%] [mg/g]
15.32 6 0.57 1.98 6 0.02 18.62 6 0.43
15.69 6 0.08 2.05 6 0.14 9.89 6 0.50
49.42 6 0.14 6.44 6 0.04 88.41 6 0.37
51.34 6 0.20 7.81 6 0.07 50.93 6 0.72
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Eur. J. Lipid Sci. Technol. 2009, 111, 135–141
500 cm3 (15.85 cm long, 12.70 cm o.d., 6.35 cm i.d.). The
holder was a rotating device with two cylinders, where the
inner cylinder (13.40 cm long, 2.54 cm in diameter) held a
400-mesh screen, while the outer one (13.40 cm long,
5.10 cm in diameter) held a 50-mesh screen. During the
experiments, the agitator was not used and the reactor
ensemble was inside a heated oven. A high-pressure pump
(Thar Technology, model P-50) with a maximum capacity of
50 g/min pumped the liquid CO2 at the desired flow rate. The
liquid cosolvent (ethanol) was pumped using a HPLC pump
(Waters; Model 510A). The CO2 or fluid mixture (CO2 1
cosolvent) was heated to the desired temperature in the pre-
reactor located inside the oven before passing through the
reactor containing the sample. The extraction was carried in a
semi-batch mode: batch charging of the sample and con-
tinuous flow of CO2 or fluid mixture. Thermocouples and
Bourdon-tube test gauges measured temperature and pres-
sure along the extraction apparatus, respectively. Pressure was
controlled by a backpressure regulator valve (BP-66; Go
Regulator Inc.). At the end of the experiment, the entire sys-
tem was depressurized and the extract was cooled in a dry-ice
bath.
2.6 Conditions of SCO2 extraction
Each experiment was done using 60 g of ground lyophilizate
placed in the sample holder inside the reactor. Experiments
were performed at a temperature of 40 7C (313 7K) and a
pressure of 300 bar (30 MPa). Such conditions above
25 MPa were shown to allow a good solubility of CoQ10 in
CO2 (.1.13610–4 mol fraction of CoQ10), which becomes
independent of the effect of temperature [35]. For each fish
specie, flow rates at 600 and 3000 g CO2/h were compared.
The latter flow rate was the highest that could be delivered by
the pumping system connected to the extractor. The flow rate
at 600 g CO2/h was compared in the presence of 5 wt-%
ethanol as cosolvent. Experiments were run during 8 h. Oil
extracts were collected after 1, 3, 5, and 8 h of extraction,
weighed, vacuum dried, reweighed and kept frozen at –80 7C
under nitrogen atmosphere until analysis. Each treatment was
repeated twice. Results were presented as cumulative yields of
oil and CoQ10, with corresponding concentrations of CoQ10
in oil, which were calculated from HPLC analysis of those
extracts.
2.7 Statistical analysis
Comparison of the performance of enzymatic hydrolysis vs.
SCO2 processes with mackerel and herring was based on
yields and recovery rates of oil and CoQ10, and correspond-
ing concentrations of CoQ10 in oil. Analysis of variance
(ANOVA) and the least significant difference test (LSD) at
p = 0.05 were done on each of these parameters.
 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Oil and coenzyme Q10 extraction from mackerel and herring 137
3 Results and discussion
3.1 Composition of fish samples used for extraction
processes
Table 1 shows that mackerel had higher lipid and CoQ10
contents but lower humidity than herring. In lyophilizates,
lipid and humidity contents were practically the same for each
fish specie, whereas CoQ10 concentration remained higher
for mackerel. Protein and ash contents were comparable for
both fish species and for both lyophilizates. However, it is
worth to mention differences of lipid-to-protein ratios
observed from each fish specie to its corresponding lyophili-
zate. For mackerel, the ratio decrease from 1.04 to 0.86 would
be explained by a lower lipid extraction efficiency on the lyo-
philizate due to the matrix effect. Adversely, a slight ratio
increase from 0.66 to 0.77 was observed for herring. The lipid
extraction method would benefit from some improvements,
like the increase of solvent-residue contact time, the number
of solvent washes, and the incorporation of water and sonica-
tion prior to lipid extraction. Such modifications were report-
ed to increase the lipid extraction efficiency on oyster lyophi-
lizates [36]. In spite of these ratio variations, lipid and protein
compositions from mackerel and herring were comparable to
those reported earlier [37, 38], which indicates the reliability
of the methods used on fresh fish samples.
3.2 Comparison of various conditions of the SCO2
process on the extraction of oil and CoQ10
Figure 1 shows the comparison of various SCO2 extraction
conditions on parameters linked to the performance of the
process, such as the cumulative yield of oil, the concentration
of CoQ10 in oil, and the cumulative yield of CoQ10, which
was calculated from the two first parameters. With mackerel
lyophilizate (Fig. 1a–c), the increase of the CO2 flow rate from
600 to 3000 g/h increased the values of all those parameters
during the extraction. When adding 5% ethanol as cosolvent
(600 g CO2/h), each parameter was still increased, and at a
quicker rate during the extraction, reaching a plateau with
maximal values after 5 h of extraction. After 8 h of extraction,
the concentrations of CoQ10 in oil reached comparable values
to those obtained without cosolvent. For herring lyophilizate
(Fig. 1d–f), the increase of the CO2 flow rate from 600 to
3000 g/h also increased the values of all the parameters during
the extraction. Adding 5% ethanol as cosolvent still increased
the cumulative yield of oil, but decreased the concentration of
CoQ10 in oil, and the cumulative yield of CoQ10. Whatever
the conditions used, the results obtained with herring gen-
erally showed lower cumulative yields of oil containing much
higher concentrations of CoQ10 and higher cumulative yields
of CoQ10, compared with mackerel. Such results could be
explained by a higher selectivity of CoQ10 extraction over that
of the oil, as the ratio of CoQ10 to lipid content was lower in
herring lyophilizate (Table 1). The increase of yield of oil with
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138 S. Laplante et al.
Figure 1. Effect of SCO2 flow rate conditions on cumulative yield of oil, concentration of CoQ10 in oil, and cumulative yield of CoQ10, with
mackerel (a–c) and herring (d–f) lyophilizates. Error bars represent standard deviations from duplicates (n = 2). The CO2 flow rate was
compared at (d) 600 g CO2/h; (m) 3000 g CO2/h; (n ) 600 g CO2/h 1 5% EtOH.
an increase of CO2 flow rate or cosolvent addition was in
agreement with earlier studies using various materials [19, 21,
39–41]. Moreover, it is worth noting that yields of oil obtained
from mackerel lyophilizates were generally higher than those
reported earlier using various moisture contents [42].
3.3 Comparison of enzymatic hydrolysis vs. SCO2
processes for oil and CoQ10 extraction from mackerel
and herring
A comparison of the performances of the enzymatic hydro-
lysis process vs. the SCO2 process (after 8 h of extraction) is
shown in Table 2. Parameters of comparison were the yield
and recovery rate (extraction efficiencies) of oil and CoQ10,
which were compared per gram of fish. The results clearly
showed higher yields and recovery rates of oil when using the
enzymatic hydrolysis process, with both fish species. The
highest values, obtained from mackerel, were comparable to
those reported using Protamex or Alcalase on various sources
of fishery by-products [22, 30].
The concentrations of CoQ10 in oil could be easily cal-
culated from data of Table 2, using yield of CoQ10 on yield
of oil. The results showed that both types of processes pro-
duced higher concentrations of CoQ10 in herring oil, espe-
cially in the case of the SCO2 process, due to a higher selec-
tivity of CoQ10 extraction over that of the oil, as explained
previously.
 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 2009, 111, 135–141
Highest yields of CoQ10 were obtained using the enzy-
matic hydrolysis process with mackerel. Highest recovery
rates of CoQ10 were obtained using this process with both fish
species, but comparable values were also observed from the
SCO2 process with herring at 3000 or 600 g CO2/h in the
presence of cosolvent (ethanol). A higher variability of yields
and recovery rates of CoQ10 was observed with herring,
especially with the SCO2 process at the flow rate of 3000 g
CO2/h, where it explained the recovery rate overestimation
above 100%. The results revealed that yields and recovery
rates of CoQ10 from herring were not much influenced by the
type of process. In contrast, for mackerel, the enzymatic
hydrolysis process was essential to significantly increase those
parameters. The higher improvements of oil and CoQ10
recovery rates obtained with mackerel would be explained by
the more important matrix effect in mackerel lyophilizate,
which limited the extraction during the SCO2 process.
The efficiency of oil extraction processes with different
types of fish species depends mainly on the composition, the
structure of the tissues and protein-lipid interactions, as
suggested by Slizyte et al. [30–32]. Lipid depots and muscle
cells (containing CoQ10) in fish are known to be usually
surrounded by a rather weak collagen network [43]. Differ-
ent fish species also contain varying amounts of collagen in
their body tissues [44–47]. It suggests that a higher collagen
content in mackerel compared with herring would explain
its higher matrix effect against CoQ10 extraction, as long as
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Eur. J. Lipid Sci. Technol. 2009, 111, 135–141 Oil and coenzyme Q10 extraction from mackerel and herring 139

Table 2. Comparison of the performances of the enzymatic hydrolysis vs. the SCO2 process for the extraction of oil and CoQ10 from
mackerel and herring.

Source of fish Type of process and conditions Oil CoQ10

Yield Recovery rate Yield Recovery rate

[g/g fish]{ [%]{ [mg/g fish]{ [%]{

Mackerel Enzymatic hydrolysis 0.135 6 0.010a 84 6 6a 15.3 6 0.5a 82 6 3ab

SCO2 600 g CO2/h 0.047 6 0.003de 30 6 2de 1.8 6 0.2e 10 6 1c
SCO2 3000 g CO2/h 0.066 6 0.007c 41 6 4cde 4.6 6 1.9de 25 6 10c
SCO2 600 g CO2/h 1 5% EtOH 0.086 6 0.005b 54 6 3b 6.1 6 0.7cd 33 6 4c
Herring Enzymatic hydrolysis 0.055 6 0.007cd 53 6 4b 8.3 6 0.7bc 84 6 6ab
SCO2 600 g CO2/h 0.025 6 0.003f 24 6 3e 6.3 6 0.8cd 64 6 9b
SCO2 3000 g CO2/h 0.036 6 0.006ef 34 6 6cde 10.3 6 2.6b 104 6 26a
SCO2 600 g CO2/h 1 5% EtOH 0.039 6 0.007ef 38 6 6cd 8.6 6 1.3bc 87 6 13ab

{
Yields from SCO2 experiments were converted from dry weight basis (lyophilizate) to fresh weight basis (fish) for comparison with the
enzymatic process. This conversion was done using the obtained ratio of 4.7483 g fish/g lyophilizate for mackerel and 5.1493 g fish/g lyo-
philizate for herring. Results were cumulative values after 8 h of extraction.
{
Recovery rates were calculated from the corresponding yield/quantity of CoQ10 or oil available per gram of fish (Table 1).
Data are means 6 standard deviation from duplicates (n = 2). Significant differences between treatments are represented by different letters
(LSD test at p = 0.05).

the enzymatic hydrolysis process is not used. Moreover, some
structural and functional protein changes were reported to
occur during SCO2 extraction of mackerel oil, such as the de-
crease of solubility and the increase of sarcoplasmic protein
aggregation [48]. Such protein changes would also contribute
to increase the matrix effect during the SCO2 process.
3.4 Economic assessment of the processes
Table 3 presents estimations of the production costs per
gram of oil and CoQ10 extracted from each type of process
under the conditions presented in Table 2. To determine
which process is more economic in view of a higher-scale
production, materials (fish, enzyme, CO2, ethanol) and lyo-
philization were the inputs considered in those estimations.
The results clearly revealed that the enzymatic hydrolysis
process generated the lowest production costs for oil and
CoQ10, which were comparable between both fish species.
This clearly revealed that the enzymatic hydrolysis process is
the most promising for up-scaling the recovery of oil and
CoQ10 from mackerel and herring. The larger scale of
production with this process and the absence of many
inputs (CO2, cosolvent, lyophilization) explained such
results. To determine which process is more economic in
view of an industrial-scale production, it is worth to mention
that many other important inputs (labor, energy, capital
investment, etc.) cannot be evaluated adequately in the
actual study. It would be more convenient to consider them
from larger-scale experiments, especially for the SCO2 pro-
cess.
Comparing the various SCO2 process conditions, oil pro-
duction costs were lower for mackerel, whereas CoQ10 pro-
duction costs were lower for herring. This was explained by
the previously shown higher yields of oil and CoQ10 obtained
from mackerel and herring, respectively. At 600 g/h CO2, the
use of cosolvent slightly decreased the oil production costs for
both fish species. It also decreased the CoQ10 production
costs for mackerel, which were increased for herring. Such
results would be another consequence of the higher matrix
effect in mackerel where the use of cosolvent would produce a
more favorable CoQ10 extraction compared to herring.
4 Conclusions
Using whole mackerel and herring, the enzymatic hydrolysis
extraction process revealed comparable or better perfor-
mance than the SCO2 process for oil and CoQ10 extraction.
Considering its lower production costs, this process would
also be a better choice in view of up-scaling the recovery of
oil and CoQ10. Future work will be oriented towards the
up-scaling and the development of fractionation processes
for enrichment of valuable lipids (polyunsaturated fatty
acids, CoQ10) found in oils extracted using the enzymatic
hydrolysis process.
Acknowledgments
The financial support from Innovation and Technologies Directo-
rate (DIT-MAPAQ), the technical assistance from the Aquatic
Products Technology Centre (CTPA), and the collaboration of Dr.
Yacine Boumghar from Centre d’Études sur les Procédés Chimi-
ques du Québec (CEPROCQ) are gratefully acknowledged.

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140 S. Laplante et al. Eur. J. Lipid Sci. Technol. 2009, 111, 135–141

Table 3. Comparison of the production costs of the oil and CoQ10 extracted using the enzymatic hydrolysis vs. the SCO2 process.

Source of Type of process and Cost of inputs used per production Production costs
fish conditions [US$]{ [US$]{

(a) (b) (c) (d) (e) (f) (g)

Fish Enzyme CO2 EtOH Lyophilization [/g oil] [/g CoQ10]

Mackerel Enzymatic hydrolysis 48.00 7.57 – – – 0.004 36

SCO2 600 g CO2/h 0.14 – 0.72 – 2.56 0.255 6667
SCO2 3000 g CO2/h 0.14 – 3.60 – 2.56 0.335 4805
SCO2 600 g CO2/h 0.14 – 0.72 1.48 2.56 0.200 2819
1 5% EtOH
Herring Enzymatic hydrolysis 24.00 7.57 – – – 0.006 38
SCO2 600 g CO2/h 0.07 – 0.72 – – 0.462 1834
SCO2 3000 g CO2/h 0.07 – 3.60 – 2.78 0.580 2027
SCO2 600 g CO2/h 0.07 – 0.72 1.48 2.78 0.419 1900
1 5% EtOH

(a) Cost of fish for enzymatic hydrolysis process = 100 kg6(0.48 $/kg mackerel or 0.24 $/kg herring). Cost of fish for SCO2 process = 0.060 kg
lyophilizate64.7483 g mackerel/g lyophilizate60.48 $/kg mackerel or 0.060 kg lyophilizate65.1493 g herring/g lyophilizate60.24 $/kg
herring.
(b) Cost of enzyme (Protamex) = 0.100 g675.72 $/kg.
(c) Cost of CO2 consumed = flow rate (g/h)68 h60.15 $/kg CO2.
(d) Cost of anhydrous ethanol consumed = 0.056total CO2 consumed66.17 $/kg anhydrous EtOH.
(e) Cost of lyophilization = 0.060 kg69 $/kg material to lyophilize.
(f) Total cost per g oil = total cost of inputs used per production/[yield oil (g/g fish)6g fish used].
(g) Total cost per mg CoQ10 = total cost of inputs per production/[yield CoQ10 (g/g fish)6g fish used].
{
Based on costs available during summer 2008.

Conflict of interest statement [7] W. N. Ratnayake, R. G. Ackman: Fatty alcohols in capelin,

The authors have declared no conflict of interest.
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