Lutein Extraction An CV
Lutein Extraction An CV
Lutein Extraction An CV
PII: S1369-703X(17)30170-5
DOI: http://dx.doi.org/doi:10.1016/j.bej.2017.06.019
Reference: BEJ 6741
Please cite this article as: Martina D’Este, Davide De Francisci, Irini Angelidaki,
Novel protocol for lutein extraction from microalga Chlorella vulgaris, Biochemical
Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.06.019
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Novel protocol for lutein extraction from microalga Chlorella vulgaris
Highlights
A novel method for the extraction of lutein from microalgae was developed.
Water was replaced by ethanol in the saponification step.
Saponification and extraction were conducted simultaneously.
Quantity of lutein extracted increased 3 folds compared to conventional methods.
Final lutein purity increased from 73.6% to 93.7%.
ABSTRACT
Lutein is a pigment generally extracted from marigold flowers. However, lutein is also
found in considerable amounts in microalgae. In this study a novel method was developed
to improve the extraction efficiency of lutein from microalga C. vulgaris. Differently from
conventional methods, ethanol was used instead of water in the saponification step, which
The amount of lutein extracted from C. vulgaris dried biomass increased more than
threefold, from 0.20 ± 0.00 mgLutein/gDM to 0.69 ± 0.08 mgLutein/gDM. Lutein purity
was increased from 73.6% to 93.7% by decreasing the ethanol-water ratio from 85% to
50% in the resolubilization step. The novel method was also tested with tetrahydrofuran.
The extraction proved to be again more effective than the conventional one; however
1
Corresponding author. E-mail address: [email protected] (D. De Francisci)
1
dichloromethane outperformed tetrahydrofuran in terms of quantity and purity of the
recovered lutein.
1. Introduction
predominant carotenoids present in the macular region of the human eye, being involved in
the protection against light-induced retinal damage and age-related macular degeneration
(AMD)[1,2]. Lutein assumption also prevents some types of cancer [3] and cardiovascular
disease [4]. Moreover, it can contribute to human bone health [5]. Lutein is also a
fundamental and an established feed additive in the poultry industry to brighten the color of
Lutein is synthesized only by photosynthetic organisms like land plants and microalgae [7].
The worldwide market for this pigment is steadily increasing, and marigold flowers are
currently the main source for the extraction and production of lutein. Microalgae have
several advantages over this flower for lutein production: first of all, a higher lutein content
and a faster growth rate [8] and the fact that they can be grown all year on infertile land and
therefore without competition with arable crops [9]. In spite of these advantages, there are
still several limitations that hinder the exploitation of this biomass for lutein production.
from this biomass into reality it is fundamental to improve and optimize the related
2
Extraction protocols from microalgae generally involve the two following separated initial
approximately 12, needed to convert lutein fatty acid esters into free lutein, 2) solvent
extraction, performed using different organic solvents combined with a cell disruption
technology [10–12].
The majority of the studies have so far focused on finding an optimal solvent for highest
extraction efficiency; a solvent that at the same time would be compatible with the
utilization of lutein for food and feed. Optimal solvents have so far being identified in
of Health and Human Services, Food and Drug Administration (FDA) [14], DCM and THF
differently from ethanol. Further steps in the extraction protocols are in fact devoted to the
Lutein extraction protocols also aim to eventually remove impurities and other unwanted
Aim of the present work was to develop an improved method for lutein extraction
compared to the conventional ones, in order to achieve higher yield and purity and, at the
same time, reduce the extraction time. In the new protocol we tested the replacement of
water with ethanol in the saponification process. Moreover, we introduced the simultaneous
saponification and extraction step. The two solvent tested were DCM and THF.
3
2.1. Chemical, reagents and biomass
All chemicals used in this work were of analytical grade and were purchased from Sigma
Aldrich ApS (Brøndby, Denmark). Lutein standard (concentration 0.804 mg/L) was obtain
ice bath. This solvent was employed due to its low specificity and therefore to obtain a
qualitative profile of the pigments present in the chosen microalgal biomass. The
supernatant was separated from the residual biomass by centrifugation at 13,000 rpm for 10
min and analyzed using a high performance liquid chromatography (HPLC) system. The
separation was achieved at 60 °C with a HPLC (Dionex UltiMate 3000, Thermo Scientific,
USA) equipped with a Zorbax Eclipse plus C8 RRHD 1.8 μm 3.0×150 mm column
(Spherisorb-ODS1 Waters, Milford, MA, USA). The mobile phase consisted of (A)
methanol/28 mM TBAA (tert-Butyl acetoacetate), pH 6.5 (70/30) and (B) methanol and the
flow rate was 0.3 ml min-1. Detection utilized UV–VIS at 450 nm. Samples and standards
were dissolved in 100% ethanol and filtered by 0.22 μm filters. After filtering a buffer was
The main steps of the conventional extraction protocol (A) are summarized in Figure 1.
4
For the saponification process to 1g of microalgal biomass were added 2.5 ml of 10 M
KOH containing 2.5% (w/v) ascorbic acid. The mixture was incubated for 10 min at 60 °C.
In the second step, 10 ml of DCM were added. The mixture was placed in a sonicator bath
for 1 hour and afterwards agitated at 300 rpm for 2 hours in an orbital shaker. After brief
vortexing the mixture was centrifuged for 5 min at 4000 rpm and the supernatant was
collected. This step was repeated until the extract was almost colorless and all extracts were
combined (in total approximately 60 mL of DCM were added). Afterwards distilled water
of the same volume as dichloromethane was added and stirred in a beaker for 4-5 minutes.
This step was performed in order to separate the lutein containing fraction from the
hydrosoluble pigments. The organic phase was then dried using a rotary evaporator, set at
40°C. The residue was redissolved in 7 ml of 85% aqueous ethanol (v/v). Hexane was
added to the mixture in the ratio 1:4 (v/v) and, after brief vortexing, was removed to
eliminate the fat soluble contaminants. Water was then added to bring the final
concentration to 8.5% ethanol (v/v) and the mixture was then centrifuged at 13000 rpm for
For each extraction process samples for HPLC analyses were taken from all the extracts
combined (60mL DCM); after the rotary evaporation step, following the addition of 85%
The main steps of the novel extraction protocol (B) are summarized in Figure 1.
For this novel protocol the saponification and the extraction steps were conducted
5
DCM were added. The mixture was placed in a sonicator bath for 1 hour and afterwards
agitated at 300 rpm for 2 hours in an orbital shaker. At this point another 2.5 mL of
ethanolic KOH 2M were added and, after brief vortexing, the mixture was centrifuged for
10 min at 4000 rpm and the supernatant was collected. At this point the extraction was
repeated by adding only DCM (in total 60 ml of DCM were used, in order to match the
quantity used in the previous protocol). The organic phase was then dried using a rotary
evaporator, set to 40°C. The residue was redissolved in 7 ml of 50% aqueous ethanol (v/v).
This solution was stirred for 10 minutes and centrifuged at 13000 g. The supernatant was
For each extraction process samples for HPLC analyses were taken from all the extracts
combined (60mL DCM); from the organic fraction after the cleaning water step; after the
rotary evaporation step, following the addition of 50% ethanol (Figure 1). The entire
Once determined that this protocol resulted in a higher extraction efficiency compared to
the conventional one and also for being less time consuming, the same procedure was
repeated replacing DCM with THF (method C) in order to compare the respective lutein
quantities.
The lutein extracts were analyzed by HPLC as described in Section 2.2. The lutein content
was detected by measuring absorbance at the wavelength range of 350–700 nm. Two
absorbance wavelengths were used (450 and 665 nm) and the maximal absorbance (450
6
Lutein was identified using Chromeleon 7.2 Chromatography Data System (CDS) software
by comparison with a library of pigments spectra. Lutein was quantified using a calibration
Purity of lutein was determined comparing the peak area of the lutein with the area of the
The lutein quantity for each sample was calculated by using the following equation:
𝑚𝑔
𝑚𝑔 𝐿𝑢𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ( 𝐿 ) × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 (𝐿)
𝐿𝑢𝑡𝑒𝑖𝑛 𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 ( )=
𝑔𝐷𝑀 𝐷𝑟𝑦 𝑐𝑒𝑙𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)
Data was analyzed with a one-way Analysis of Variance (ANOVA) followed by Tukey’s
test (p < 0.05) to evaluate if there were significant differences among the results obtained
for different extracts. The software used to carry out the statistical analyses was
Figure 2 shows the pigment composition profile of the C. vulgaris biomass tested in this
study. The chromatogram shows high heterogeneity of the hydrophobic pigments, with the
most abundant ones being lutein (Figure 2, peak 3), chlorophyll a (Figure 2, peak 4) and
chlorophyll b (Figure 2, peak 5). Therefore, a procedure to selectively extract and purify
lutein is needed.
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3.2 Summary of the differences between the conventional and the novel
methods
A novel protocol was developed and tested on C. vulgaris biomass. In this protocol, water
was replaced with ethanol in the saponification process, based on the assumption that,
being lutein insoluble in water and 100% soluble in ethanol, employing this solvent would
minimize partial precipitation of the pigment in this specific step. The saponification and
the extraction steps were also combined in order to simplify the entire process as it was
done for marigold flowers [15,16]. The validity of this improved protocol was assessed by
performing the extraction both with the conventional extraction method (saponification
with aqueous KOH and separated from the solvent extraction) and the one developed in this
work, and eventually comparing the results. DCM was employed as solvent for this
comparison as it was the one most widely used for to extraction of lutein from microalgae.
After having proved that these modifications hugely improved the conventional protocol as
hypothesized, THF was also tested in the same optimized conditions in order to determine
whether this solvent could outcompete DCM as the elective organic solvent for the
The differences in the initial steps of the two extraction protocols (i.e., conventional method
and novel method) are summarized in Figure 1. The chromatographic analyses (Figure 3)
showed that, due to the introduced modifications but most probably mainly for the
remarkable 3 folds, from 0.31 ± 0.06 mgLutein/gDM (conventional method) to 1.00 ± 0.15
8
(Table 1, step 1). Most probably the reason of such an increase is imputable to the different
solubility of lutein in water and ethanol (0 and 100% respectively). The utilization of
ethanol instead of water in this critical step has probably resulted into a higher conversion
of lutein fatty acid esters into free lutein and this despite the much lower concentration of
KOH that guaranteed anyway the maintenance of pH at 12 for the entire process. In both
cases the purity of lutein amounted to approximately 70% due to the presence in the extract
The final quantity of lutein achieved using protocol B was 0.69 ± 0.08 mgLutein/gDM,
more than 3 times the amount achieved with protocol A (0.20± 0.00 mgLutein/gDM). All
the results are summarized in Table 1 and can be visualized in Figure 5. It is also
noteworthy that in the conventional protocol more lutein is lost through the different
purification steps compared to protocol B (Table 1). The rotary evaporator step was used to
completely remove the DCM before resolubilizing the dried pigments mixture, mainly
DCM is due to its toxic nature even in remote traces, which is inappropriate in drug and
food products [14]. This step was also necessary for increasing the purity of lutein. Indeed,
chemical properties of lutein to selectively precipitate this pigment while keeping the others
in solution. Lutein being completely insoluble in water, maximizing the ethanol in the
resolubilization step enables a higher lutein recovery. Our results show (Figure 4) that a
9
50% ethanol-water ratio translates into a much higher lutein purity compared to 8.5% ratio
In Figure 4 is possible to notice that neoxanthin and violaxanthin were almost completely
removed from the mixture. Another important alteration in comparison with the
conventional extraction method is the removal of the hexane step from the protocol. This is
an additional advantage of the novel protocol as hexane is also considered toxic for food
and drugs.
quantity and quality of lutein recovered, the same protocol was tested using THF instead of
DCM. THF was chosen because this solvent was proven to be compatible for the
solubilization and purification of lutein from microalgae in previous works [13]. The
results showed that DCM was still the solvent of election for this process as protocol B
0.00 mgLutein/gDM of THF (Table 1, step 3 and Figure 5). Moreover protocol B achieves
a higher purity than protocol C (93.7 % and 87.4% respectively) (Table 1).
In literature, the total lutein content of microalgae biomasses are calculated using extraction
protocols whit diethyl ether or DCM as solvent [10,13]. This specific value is necessary to
calculate the final extraction yield. In Chan et al. the quantity is extracted with DCM and
diethyl ether is not significantly different. In Chen et al. THF is found to outperform diethyl
ether. None of these methods utilizes ethanol in the saponification step which, according to
10
our results and to a vast number of papers in which lutein is extracted from Marigold
flowers [17,18], increases remarkably the solvent performance. Indeed, the higher lutein
solubility in ethanol enables a lower lutein precipitation in the saponification step, with a
consequent higher yield. Furthermore, always according to our results, DCM outperforms
THF significantly. Based on all these considerations it was concluded that the amount of
lutein extracted from the first step of protocol B (DCM as extraction solvent and ethanol in
the saponification process) is the most accurate and therefore the one to be considered as
total lutein content of this specific biomass (1.00 ± 0.15 mg/gDM). Based on this
assumption the final yields resulted to be 20, 69 and 41% for protocol A, B and C,
reported in the study of Lin et al. (2015), a lutein productivity of 0.11, 0.37 and 0.22 g/L/d
Conclusion
In this work a novel protocol for the extraction of lutein from microalgae was developed. In
the saponification step water was replaced with ethanol to minimize loss due to
conventional protocols, both when using DCM or THF as extraction solvent. The use of
Acknowledgements
This work resulted from the BONUS Microalgae project was supported by BONUS (Art
185), funded jointly by the EU and the Danish Agency for Science, Technology and
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Innovation (DASTI), the Estonian Environmental Investment Centre (KIK) and The
We thank Satomi Matsuura and Mikael Emil Olsson for the technical support.
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Figure captions
methods. Only the main steps are included. Samples for HPLC analyses were taken from the steps
Figure 3: Chromatogram of crude lutein obtained by extraction with DCM with the conventional
method (a) and with the novel one (b). Peaks: 1, neoxanthin; 2, violaxanthin; 3, lutein.
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Figure 4: Chromatogram of purified lutein obtained with DCM with the conventional method (a)
and with the novel one (b). Peaks: 1, neoxanthin; 2, violaxanthin; 3, lutein.
Figure 5: Chromatogram of purified lutein obtained with the novel method using DCM (a) or THF
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Figr-1
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Figr-2
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Figr-3
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Figr-4
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Figr-5
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Table 1: Lutein extracted (mggDM-1) and purity through the different purification steps in protocol A, B and C.
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