Abstract:
This work presents an original approach to develop
an integrated process to improve
the nutritional characteristics of natural oils, starting with the
extraction from the raw material by environmentally friendly methods and
following with the production of novel acylglycerols using immobilized
lipases. Specifically,
2‑monoacylglycerols (2-MAGs)
enriched in the omega-3 stearidonic acid (SDA) were synthesized by
selective ethanolysis of extracted Echium plantagineum oil
using the lipase fromThermomyces lanuginosus (TLL). Different reaction conditions were
investigated to minimize the undesirable acyl migration and to ensure
the purity of final products. The biocatalyst produced in our laboratory
by the immobilization of TLL on a hydrophobic support reached the
maximum theoretical amount of 2-MAG in only 2 h at mild reaction
conditions, achieving a product
enriched in omega‑3 SDA (up to
25%). Moreover, the produced biocatalyst
exhibited higher stability than
commercial lipases. The average activity after 5 cycles was 71%,
allowing several reutilization cycles and developing a feasible
enzymatic process. Finally, 2‑MAGs was used as starting material to
synthesize structured triacyclglycerols (STAGs) in solvent-free systems.
The use of molecular sieves in
combination with the immobilized lipase from Rhizomucor miehei(RML) showed to be an extraordinarily fast strategy to produce pure
STAGs (100% in 1h), 4 times higher than the activity showed by the
commercial derivative. Thus, the enzymatic processes developed in this
study open a range of possibilities to synthesize omega-3 acylglycerols
with improved characteristics for essential biological functions and
nutritional advantages, proving
the usefulness of immobilized
lipases to produce novel functional lipids.
Keywords: omega-3 PUFAs, enzymatic modification, lipase
immobilization, green process, bioactive lipids, Echium
plantagineum L.
Introduction
In recent years, interest in omega-3 polyunsaturated fatty acids
(omega-3 PUFAs) has increased because of their numerous functions in
promoting human health. Omega-3 PUFAs have been associated with a lower
risk of cardiovascular diseases, inflammation, cancer and neurological
disorders (Bowen et al., 2016; Shahidi and Ambigaipalan, 2018). The
omega‑3 α-linolenic acid (ALA, 18:3 all cis-9,12,15) is considered an
essential fatty acid from which all other omega-3 PUFAs are
metabolically derived. ALA can be converted into stearidonic acid (SDA,
18:4 all cis-6,9,12,15), eicosapentaenoic acid (EPA, 20:5 all
cis-5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6 all
cis-4,7,10,13,16,19) in the human body by elongation and desaturation
pathways, but their conversion is often poor (less than 8%). This poor
conversion can be explained by the initial Δ6 desaturase enzyme being
rate limiting in humans. However, SDA is more efficiently converted to
EPA than ALA because does not require the first of the rate‑limiting
step (Walker et al., 2013). SDA has been found to be further metabolizedin vivo and lead to increase the conversion into EPA with
approximately 20–26% efficiency (Brenna et al., 2009). Hence, SDA
provide a superior strategy for the biosynthesis of omega-3 very
long-chain polyunsaturated fatty acids (omega-3 VLC‑PUFAs) in the human
body.
On the other hand, the form of administration of omega-3 PUFAs is a
crucial factor, with significant differences between different lipid
classes in bioavailability, absorption, metabolism and bioactivity in
the human body (Castejón and Señoráns, 2020). In natural sources,
omega-3 PUFAs are primarily present as triacyclglycerols (TAGs) and, to
a lesser extent, as free fatty acids (FFAs), phospholipids (PLs) or
other acylglycerols. It should be highlighted that the position at which
the omega-3 PUFAs are attached to TAGs plays an important role in their
absorption. Omega-3 PUFAs attached to the sn -2 position are
preferentially absorbed as monoacylglycerols (MAGs) after the cleavage
of fatty acids from sn -1 and sn -3 positions by pancreatic
lipases, whereas the chain length and number of double bonds of
resulting FFAs will influence its absorption. Unsaturated fatty acids
and short- and medium‑chain fatty acids (SCFAs and MCFAs) are more
efficiently absorbed than VLC-PUFAs. Moreover, the presence of calcium
and other ions can reduce the bioavailability of FFA by forming
insoluble complex. Based on these findings, trends in food science and
biotechnology have focused on the synthesis of novel functional lipids
by modifying the composition and/or distribution of fatty acids in the
glycerol backbone, aiming at desirable nutritional properties and
improving their bioavailability.
Lipases are a versatile class of enzymes that catalyze a broad variety
of reactions, being the most stablished biocatalyst used in lipid
modification (Bornscheuer, 2018). Enzymes offer a range of advantages
over chemical methods, such as high chemo-, regio-, and
stereoselectivity, mild reaction conditions and low temperatures,
minimal undesirable by‑products, and less omega-3 PUFAs oxidation
(Castejón et al., 2019). Moreover, the use of enzymes is considered an
environmentally friendly process because enzymes are biodegradable,
produce minimal waste and allow to use water or other green solvents as
reaction medium (Jegannathan and Nielsen, 2013). Nevertheless, lipases,
as most enzymes, need to be immobilized for their applications as
biocatalysts to improve their stability and facilitate their separation
from the reaction medium as well as enzyme reuse (Mateo et al., 2007).
The development of stable and active biocatalyst with the possibility of
using it for several cycles could significantly reduce process costs. In
this sense, the immobilization of lipases via interfacial activation on
hydrophobic supports has been reported to be an efficient method to
immobilize lipases. This method fixes the open form of lipases via
interactions between the hydrophobic surroundings of their active centre
and the hydrophobic surface of the support (Fernandez-Lorente et al.,
2008). Biocatalysts prepared by following this immobilization mechanism
have shown enhanced activity against hydrophobic substrates and improved
stability under different experimental conditions (Fernández-Lorente et
al., 2011).
In this work, enzymatic production of 2-MAGs enriched in omega-3 SDA was
studied by ethanolysis using the lipase from Thermomyces
lanuginosus (TLL) immobilized on a hydrophobic support in our
laboratory. To develop an integrated process, in a first step, omega-3
oil was extracted from the highest vegetable source of SDA, Echium
plantagineum L., by an eco-friendly method using pressurized liquids
and ethyl acetate as solvent. In a second step, to minimize the
undesirable acyl migration and to ensure the purity of final products,
different reaction conditions were investigated: time, temperature and
type of biocatalyst. Reaction products were analyzed by HPLC-ELSD and
GC-MS to evaluate the fatty acid composition of synthesized 2‑MAGs.
Furthermore, reutilization of produced biocatalyst was studied to assess
the effect of immobilization in the stability of lipase derivatives.
Finally, to show the potential application of synthesized products,
2-MAGs was used as starting material for the synthesis of structured
triacyclglycerols (STAGs). Thus, the main objective of the present work
was to develop an efficient and integrated process for the synthesis of
omega-3 acylglycerols with high added‑value using immobilized lipases
for potential applications in food and nutraceutical industries.
- Materials and methods
- Materials.
Echium seeds (Echium plantagineum L.) were provided by Technology
Crops International (Essex, United Kingdom). Seeds were ground with a
particle size less than 500 μm using a grinder (Moulinex-A320R1 700 W)
and stored at 4 °C until the oil extraction process.
Soluble lipases from Thermomyces lanuginosus and Rhizomucor
miehei and commercial derivative Lipozyme TL IM and Novozym 40086 were
kindly donated for Novozymes (Bagsvaerd, Denmark). Sepabeads-C18 was
kindly donated by Resindion S.R.L. (Rome, Italy). P‑nitrophenyl butyrate
(pNPB) were provided by Sigma Chemical Co. (St. Louis, USA). Absolute
ethanol (PRS grade), sodium hydrogen carbonate and potassium hydroxide
were purchased from Panreac Quimica S.A (Barcelona, Spain). Caprylic
acid ethyl esters were purchased from Sigma-Aldrich Co. LLC (Darmstad,
Germany). Solid phase extraction cartridges (Bond Elut NH2) were from
Agilent (Palo Alto, CA, USA). Molecular sieves pore size 4 Å
(pearl-shaped 2–3 mm) and n-hexane was purchased from Scharlau
(Barcelona, Spain). The solvents (2,2,4-trimethyl pentane, methyl
tert-butyl ether and 2-propanol) used for high-performance liquid
chromatography (HPLC) analyses were HPLC-grade and purchased from
LABSCAN (Dublin, Ireland). Fatty acid methyl esters standard (Supelco 37
FAME Mix) was from Supelco (Bellefonte, PA, USA). Glyceryl trilinoleate,
dioleoylglycerol (mixture of 1,3- and 1,2-isomers),
1-oleoyl-rac-glycerol, oleic acid and ethyl linoleate used as HPLC
standards was purchased from Sigma-Aldrich (St. Louis, MO, USA). All
other reagents and solvents used were of analytical or HPLC grade.
Pressurized liquid extraction of Echium plantagineum seed oil
Pressurized liquid extraction (PLE) was carried out with an ASE 350
DIONEX (Sunnyvale, California) extractor. Oil extraction was performed
using 3 g of ground echium seeds. Stainless steel extraction cells were
used with a capacity of 10 mL. Extracts were collected under a nitrogen
stream in different vials of 50 mL. Extraction conditions used were
performed according to previous studies using ethyl acetate as solvent
at 150 °C and 10 min of static time (Castejón et al., 2018).
The samples were evaporated in a rotary evaporator (Heidolph Hei-Vap
Value HB/G3, Germany) under reduced pressure at 40 °C and dried under a
nitrogen stream until constant weight. The oil content was determined
gravimetrically and expressed as dry weight percentage. Oil obtained was
stored in dark vessels with nitrogen atmosphere at 4 °C until their use.
Immobilization of lipases on Sepabeads-C18 resins
Lipase from Thermomyces lanuginosus and lipase fromRhizomucor miehei were immobilized on Sepabeads-C18 resins by
hydrophobic adsorption at low ionic strength (5 mM) in sodium phosphate
buffer at 25 °C and pH 7. To follow the immobilization process, the
activity of the blank and the supernatant were analyzed
spectrophotometrically at different times by measuring the absorbance at
348 nm (∈ = 5.150 M−1 cm−1) produced
by the release of p-nitrophenol (pNP) by hydrolysis of 0.4 mM
p-nitrophenyl butyrate (pNPB) in 25 mM sodium phosphate buffer at pH 7
and 25 °C. After 24 hours, enzyme loading of lipase derivatives was 33.0
mg of TLL per gram of support and 29.6 mg of RML per gram of support.
Lipase derivatives were washed and dried, before their use in enzymatic
reactions, with increasing volumes of water, water: acetone and acetone
using a sintered glass funnel until derivatives were completely dried.
Enzymatic synthesis of 2-MAGs from echium oil
Enzymatic synthesis of 2-MAGs was performed by ethanolysis reaction. In
a typical experiment, echium oil and ethanol (ratio 1:4 (w/w)) and 15%
(w/w) of the biocatalyst Lipozyme TL IM were place in glass vials. The
reaction was carried at 25 °C and 35 °C with constant stirring in an
orbital shaker at 200 rpm (Unimax 1010, Heidolph, Germany) under dark
conditions. A negative control without biocatalyst was carried out at
the same conditions previously described. The reaction mixture was
filtered to remove the biocatalyst and the excess of ethanol was removed
under vacuum. The synthesis of 2‑MAGs was followed by HPLC-ELSD.
Reaction kinetics were done at least in duplicate. 2-MAGs obtained were
stored in dark vessels with nitrogen atmosphere at 4 °C until their use.
Transesterification between 2-MAGs and caprylic acid ethyl esters
Transesterification between the synthesized 2‑MAGs and caprylic acid
ethyl esters was performed to produce STAGs as previously described
(Castejón and Señoráns, 2019). In a typical experiment, ethanolysis
reaction product (2‑MAGs) and caprylic acid ethyl esters ratio 1:20
(w/w), 10% (w/w) of biocatalyst and molecular sieves were place in
glass vials. The reaction was carried out at 35 °C with constant
stirring in an orbital shaker at 200 rpm (Unimax 1010, Heidolph,
Germany) under dark conditions. A negative control without biocatalyst
was carried out at the same conditions previously described. The
synthesis of STAGs was followed by HPLC ELSD. Reaction kinetics were
done at least in duplicate.
Reutilization cycles of lipase derivatives
For recycling studies, the enzymatic reaction was carried out in
PP-Reactors, 10ml, with PTFE frit (Multisyntech GmbH, Witten, Germany).
After the optimal reaction time, reaction volume was carefully
filtrated, lipase derivatives were washed with 1 mL of hexane and
separated from the reaction medium. A new batch with the reutilized
lipase was carried out at the same conditions previously described until
lipases totally lost their activity. The 2-MAG yield of the first
reaction was set as 100% and the yield in the subsequent reactions was
calculated accordingly.
Lipid fractionation by Solid Phase Extraction
Reaction products were fractionated using solid phase extraction (SPE)
with ISOLUTE® NH2 columns (aminopropyl bonded sorbent) as previously
described (Castejón and Señoráns, 2019). SPE columns were preconditioned
with 4 mL of hexane. The sample (50 mg reaction product/500 mL hexane)
was allowed to adsorb to the matrix by percolation through the cartridge
by gravity. SPE column was eluted with 10 mL of solvent A (hexane), 4 mL
of solvent B (hexane: isopropanol (10:1)) and 4 mL of solvent C (hexane:
isopropanol (3:1)). The fractions eluted from the SPE column were dried
down under nitrogen and re-dissolved to analysis by HPLC-ELSD. Fatty
acid ethyl esters (FAEEs) were eluted in fraction 1, TAGs and
diacylglycerols (DAGs) in fraction 2, and 2-MAGs in fraction 3.
HPLC-ELSD analysis
The lipid composition as the enzymatic reaction time proceeded was
kinetically controlled and analyzed by HPLC-ELSD. Time reaction samples
were taken after 0, 0.3, 0.7, 1, 2, 3, 4, 6 and 8 h. Samples were stored
at -20 °C until their analysis.
HPLC-ELSD analyses were performed using an Agilent 1260 Infinity HPLC
equipped with an Agilent 385 (Palo Alto, CA, USA) ELSD instrument. The
chromatographic separation of FAEE, TAGs, DAGs and MAGs was performed
with a silica normal-phase ACE (250 mm x 4.6 mm i.d., 5 µm) column
maintained at 30 °C using a ternary gradient as follows: 0–2 min,
99.5% A and 0.5% B; at t = 6.5 min, 70% A and 30% B; at t = 11 min,
63% A, 27% B and 10% C; at t = 18 min, 99.5% A and 0.5% B; and at t
= 20 min, 99.5% A and 0.5% B. Eluent A consisted of 2,2,4
trimetilpentane, eluent B consisted of methyl tert-butyl ether, and
eluent C consisted of 2-propanol. The flow rate was 2.0 mL/min except
for minutes 13 to 16 which was 1.0 mL/min. Optimal signal and resolution
were achieved with the following ELSD conditions: evaporator temperature
= 30 °C; nebulizer temperature = 30 °C; and evaporator gas N2 = 1.6 SLM.
Fatty acids composition by GC-MS
Fatty acids composition of echium oil and reaction products were
analyzed on an Agilent GC MS series 5975 MSD (Palo Alto, Cal., USA)
using a HP 88 capillary column (100 m x 0.25 mm, i.d. 0.2 µm) (Agilent,
CA, USA). Previous to analysis, fatty acid methyl esters (FAMEs) were
prepared by base-catalyzed methanolysis of the echium glycerides (KOH in
methanol). 1 µL sample was injected using a split ratio of 1:100. The
column was held at 175 °C for 10 min after injection, the temperature
programmed at 3 °C/min to 220 °C and held for 20 minutes more. Helium
was used as gas carrier, at a constant column flow rate of 1.5 ml/min.
The injector temperature was 250 °C and the detector temperature was 230
°C. The mass spectrometer was operated at 70 eV with a mass range from
30 to 400 amu. Fatty acids were identified comparing their retention
times and the mass spectra (NIST MassSpectral Library Version 2.0) with
those obtained from the standards.
- Results and discussion
- Pressurized liquid extraction of Echium plantagineum seed oil
Echium oil was extracted from Echium plantagineum seeds by a
green method using pressurized liquids. Given the fact that the
development of faster, less solvent consuming, full automation and more
environmentally friendly extraction methods has grown in importance
during the last decade, the use of PLE has become a consolidated
extraction technique. Additionally, to follow the principles of green
chemistry and to avoid hazardous and toxic solvents, ethyl acetate was
selected to carry out the extraction process. The maximum oil yield was
achieved at the temperature of 150 °C (31.0% ± 0.7). Results
were compared with an oil extraction reference solvent, hexane,
achieving similar oil yield (31.1% ± 0.3) (no significant differences
at 1% level). Therefore, echium oil to produce 2‑MAGs was freshly
extracted using PLE with ethyl acetate, providing an overall extraction
process based on eco-friendly approaches.
Enzymatic synthesis of 2-MAGs from echium oil: process optimization
Enzymatic ethanolysis of TAGs is an effective method to produce 2-MAGs
from natural sources. Even though, a key factor implied on the success
of this reaction is the selection of the type of lipase to be used. In
this study, the lipase from Thermomyces lanuginosus was selected
due to the high regioselectivity towards the positions sn -1 andsn -3 of the glycerol backbone (Fernandez-Lafuente, 2010),
producing 2‑MAGs from echium oil by selective hydrolysis.
In a first stage, reaction conditions were optimized to produce the
maximum efficiency yield in the shorter reaction time. Specifically, a
comparison between commercial TLL from Novozyme (Lipozyme TL IM) and a
biocatalyst produced in our laboratory (soluble TLL immobilized by
hydrophobic adsorption on Sepabeads C-18) was carried out. To minimize
the undesirable acyl migration, which can take place during hydrolysis
of TAGs, different reaction conditions were investigated. Acyl migration
in the glycerol backbone often leads to the increase of by-products that
could significantly affect the purity of final products, like 1‑MAGs
that could easily hydrolyze due to the specificity of lipase used. The
effect of reaction conditions on acyl migration has been elucidated and
temperature is one of the most important factors that have a deep
influence on the migration rate, since acyl migration is a thermodynamic
process (Yang et al., 2005). Thus, in this work, ethanolysis reaction
was carried out at mild reaction conditions studying two temperatures,
25 °C and 35 °C. Another critical factor in acyl migration rate is the
polarity of the solvent used as reaction medium. Several authors
described the synthesis of 2-MAGs by ethanolysis using acetone (Muñío et
al., 2009; Pfeffer et al., 2007) or hexane (Wang et al., 2014) as
reaction medium. However, Li et al. concluded that solvent polarity is a
crucial factor for acyl migration: decreasing solvent polarity would
increase acyl migration rate (Li et al., 2010). Thus, polar solvents are
highly recommended for this reaction. In the present work, ethanolysis
was carried out in pure ethanol without the use of an additional solvent
as reaction medium. The advantage of using ethanol, besides the
minimization of acyl migration, lies in the fact that ethanol is
considered a green solvent allowed in the food industry.
Figure 1 shows 2-MAGs percentage based on maximum theoretical yield of
total hydrolysis products (maximum theoretical yield = 33% of initial
oil TAGs) identified by HPLC-ELSD at the investigated temperatures
during reaction progress. After 20 min, the commercial biocatalyst
reached the maximum theoretical yield at 25 °C (100%), while the same
reaction at 35 °C showed a lower production yield (74.2%). After 1 h,
commercial lipase did not show differences between tested temperatures.
However, a different behavior was found for TLL adsorbed on
Sepabeads C‑18, where the initial hydrolysis rate of the immobilized
biocatalyst was lower. For instance, at the temperature of 25°C,
ethanolysis reaction gave a yield of 20% after 20 min, but after 1 h,
2-MAGs yield was 89.9%. According to these results, TLL immobilized on
Sepabeads C‑18 needs more time to reach the maximum theoretical yield
(see further discussion). Furthermore, the effect of temperature on the
synthesis of 2‑MAGs for TLL adsorbed on Sepabeads C‑18 was more
outstanding in comparison with the commercial derivative. After 1 h,
2‑MAGs yield was 27.5% at 35 °C, around 3 times less than the yield
reached at 25 °C. Therefore, the optimum temperature to produce 2-MAGs
from echium oil was established at 25 ° C.
To fully evaluate and understand the synthesis of 2-MAGs, it is also
necessary to analyze the lipid composition of the reaction mixture as
the reaction time proceed, including TAGs, DAGs and 2‑MAGs. Moreover, to
select the optimum reaction time for the synthesis of 2‑MAGs, in this
work, different requirements were stablished: TAGs need to be completely
hydrolyzed and DAGs need to be less than 5% of the total reaction
products. For this purpose, Figure 2 shows the lipid composition
analyzed by HPLC-ELSD as the reaction time proceed catalyzed by tested
biocatalysts at 25 °C. As can be seen in Figure 2(a), TAGs were almost
completely hydrolyzed by TLL commercial after 20 min, and the reaction
mixture was composed by 50% FAEEs (data not show in Figure 2), 1%
TAGs, 16% DAGs and 33% of 2‑MAGs. After 2 h, commercial lipase
hydrolyzed DAGs below 5%.
On the other hand, TLL adsorbed on Sepabeads C-18 (Figure 2(b))
exhibited lower initial activity in comparison with the commercial
biocatalyst. This fact could be due to the different enzyme loading in
terms of amount of lipase per reaction volume. Since the enzymatic load
is not specified in the commercial biocatalyst and in order to minimize
this effect, the amount of TLL adsorbed on Sepabeads C-18 required to
achieve the same TAGs hydrolytic rate produce by the commercial
biocatalyst after 20 min was calculated. As a result, the reaction
kinetic of TLL adsorbed on Sepabeads C-18 using equivalent enzyme
loading is shown in Figure 2(c). As expected, the use of a higher enzyme
loading implied a high initial velocity for the ethanolysis reaction.
After 40 min, the theoretical percentage of 2-MAGs was reached and after
2 h, DAGs were less than 5%. Thus, the optimal time for production of
2-MAGs from echium oil was established at 2 h.
Other authors reported the synthesis of 2-MAGs from echium oil using
pancreatic lipase, achieving a 2-MAGs yield of 79.1% (percentage
expressed as maximum theoretical yield, 33% of total reaction products)
(Rincón Cervera et al., 2013). However, the production of 2‑MAGs using
pancreatic lipase is not recommended if the reaction products are later
to be used in the synthesis of structured lipids, since water residues
could favor the hydrolytic activity of lipases, carrying out the
opposite reaction.
Fractionation and characterization of synthesized 2-MAGs by GC-MS
One of the objectives of the present study was to evaluate if the
synthesized 2-MAGs were enriched in the omega-3 SDA. Consequently, once
reaction conditions and optimal reaction time were established to
synthesize 2‑MAGs, reaction products from echium oil were analyzed by
GC-MS to evaluate the fatty acid composition. First, the isolation of
2-MAGs from the reaction mixture was done by SPE using a polar
stationary phase. Three fractionation steps were necessary to remove the
excess of FAEE and to achieve a pure fraction of 2-MAGs. SPE fractions
were analyzed by HPLC‑ELSD, an example is shown in chromatogram in
Figure 3. As can be seen, the fractionation process was successfully
done. Figure 3(b) shows the reaction mixture before fractionation
process (original reaction products), which were composed by 2-MAGs and
FAEEs. Finally, after the fractionation process by SPE, a pure fraction
of 2‑MAGs was achieved (Figure 3(d)).
The purified 2-MAGs were analyzed by GC‑MS to determine the fatty acids
content and compare it with the original oil. Moreover, the effect of
biocatalyst type on the fatty acid composition was also studied. Table 1
shows the results of the GC-MS analysis. Fatty acid composition (% of
total fatty acids) of original echium oil in growing order of abundance
was stearic acid (18:0), palmitic acid (16:0), γ-linolenic acid (18:3
all-cis-6,9,12), linoleic acid (18:2 cis-cis-9,12), stearidonic acid
(18:4 all cis-6,9,12,15), oleic acid (18:1 cis-9) and α-linolenic acid
(18:3 all-cis-9,12,15). Echium oil was characterized by a low percentage
of saturated fatty acids (SFA) (10.2%) and monounsaturated fatty acids
(MUFA) (16.8%), while the percentage of polyunsaturated fatty acids
(PUFA) (73.0%) was the highest of total fatty acids. Regarding to the
omega-3 content, ALA and SDA were identified with a percentage of 35.0%
and 15.5%, respectively. Results were in accordance with other authors
(Surette, 2013) and previous studies of the research group (Castejón et
al., 2018).
On the other hand, synthesized 2-MAG was composed by oleic acid,
linoleic acid, γ‑linolenic acid, ALA and SDA, being the omega-3 ALA and
SDA the major fatty acids. Specifically, the omega‑3 SDA was identified
in a percentage of 25%, implying than produced 2-MAGs was enriched in
the omega-3 SDA 1.6 times compared with the original oil. In addition,
the effect of the type of lipase derivative used in the omega-3
composition of 2‑MAGs was studied. 2‑MAGs synthesized by TLL absorbed on
Sepabeads C-18 was characterized by a SDA percentage of 25.5% ± 0.1,
similar percentage were identified in 2‑MAGs produced by commercial
biocatalyst (25.0% ± 0.3) (no significant differences at 1% level).
These results demonstrated that the fatty acid composition was not
modified regarding the type of lipase used.
Therefore, the enzymatic production of 2-MAGs under mild reaction
conditions and low temperature (25 °C) managed to enrich the omega-3
SDA, obtaining an attractive starting material to synthesize structured
lipids with potential interest as functional ingredients. Other authors
have investigated the enrichment of stearidonic acid from echium oil,
however, following a complex process via two-step lipase-catalyzed
esterification (Baik et al., 2014; Baik et al., 2015). These results
show the ability of enzymatic modification in the enrichment of specific
fatty acids such as stearidonic acid.
Stability of lipase derivatives in the synthesis of 2-MAGs from echium
oil
Enzyme stability is a crucial factor to determine whether the
application of biocatalysts will be commercially successful.
Reutilization of biocatalyst produced in our laboratory and commercial
derivative in the synthesis of 2-MAGs from echium oil was studied to
evaluate the effect of immobilization in the stability of lipase
derivatives.
2-MAGs percentage synthesized at 2 h of each lipase derivative was taken
as reference value (100%), subsequent reaction yields were calculated
accordingly. As can be seen in Figure 4, commercial derivatives had
serious limitations for reuse in this reaction. TLL commercial lost 25%
of its activity in the second cycle and 70% in the fourth cycle.
However, the reuse of TLL adsorbed on Sepabeads C-18 was possible. In
this case, after the second cycle the percentage of reuse was 92%, and
after five reaction cycles, the average activity of the immobilized
lipase was 71%. These results suggest that the immobilization process
by hydrophobic interactions carried out for the lipase ofThermomyces lanuginosus improves the stability of the produced
biocatalyst in comparison with commercial derivatives. Thus,
biocatalysts developed in our laboratory seem to have reasonable
industrial possibilities, since the lipases can be reused for several
cycles in ethanolysis reaction with a little loss of activity.
Enzymatic transesterification of 2-MAG with caprylic acid ethyl esters
In a first approach to synthesize structured triglycerides and to show
the potential application of synthesized products, transesterification
between the produced 2-MAGs from echium oil with caprylic acid ethyl
esters was carried out. The incorporation of short- and/or medium‑chain
fatty acids into TAGs is the most representative and well-known example
of structured lipid. The advantage of using short- and medium‑chain
fatty acids relies on the fact that these fatty acids are rapidly
absorbed into the bloodstream by the intestinal capillaries and
efficiently converted into energy, unlike long chain fatty acids, which
require bile salts for digestion. Moreover, short- and medium‑chain
fatty acids are more accessible to lipases due to their small size. This
strategy has been used for the synthesis of STAGs with different
nutritional purposes (Korma et al., 2018; Utama et al., 2019). For
instance, these STAGs included essential long-chain fatty acids located
at sn -2 and short- and medium‑chain fatty acids located
at sn ‑1,3. Specifically, the STAG synthesized in this study was
composed by the omega-3 stearidonic acid located at sn -2 and
caprylic acid located at sn -1,3.
As it is known, under favorable conditions, lipases can catalyze
esterification reactions, but it is important to control the reaction
medium, since these reactions are usually reversible. In the
transesterification reaction proposed in the present study, the ethanol
produced during esterification must be removed from the medium since it
can act as a catalyst for the hydrolysis reaction. For that reason, it
is necessary to shift the equilibrium to favor the formation of desired
products. The most effective strategies for this purpose are: the
application of high temperatures or vacuum, induce the change of state
of the product to be eliminated, the use of membrane technology
(pervaporation) or the use of molecular sieves as adsorbing agents.
In this study, the use of molecular sieves in transesterification
reaction was evaluated by comparing the lipase from Rhizomucor
miehei immobilized on Sepabeads C-18 in our laboratory and the
commercial derivative Novozym 40086. The selection of RML was based on
lipase specificity and previous unpublished results from the research
group. Moreover, to continue the efforts to promote the development of
eco-friendly alternatives, the reaction was carried out in a
solvent-free system, acting ethyl esters themselves as reaction medium.
Figure 5 shows the reaction kinetic of transesterification between
synthesized 2-MAGs and caprylic acid ethyl esters. Furthermore, Figure 6
shows an example of chromatograms corresponding to the HPLC-ELSD
analysis at initial time, after 20 min and after 1 h of reaction.
It is noteworthy that the synthesis rate was extraordinarily fast for
this reaction, STAGs could be observed after only 20 min (Figure 6.b)
and, after 1 h, the reaction was completed, identifying only an excess
of FAEEs and the targeted reaction product (Figure 6.c). In contrast
with previous results reached in the production of 2-MAGs, lipase
adsorbed on Sepabeads C‑18 exhibited higher initial activity than the
commercial derivative. After 20 min, RML adsorbed achieved a percentage
yield of 64% in comparison with 2.9% reached by the commercial
biocatalyst. Moreover, the reaction was complete in 1 h by RML adsorbed
on Sepabeads C-18, while the commercial lipase did not reach the 100%
of STAGs until 4 h. Therefore, the synthesis activity exhibited by RML
adsorbed on Sepabeads C-18 was 4 times higher than the activity showed
by the commercial derivative. This result suggest that the type of
reaction also influences on the efficiency of each lipase derivative on
the same support.
Other authors have been described the synthesis of structured lipids
from echium oil containing short- and/or medium‑chain fatty acids but
following other strategies. For example, using free fatty acids from
echium oil and tricaprylin by enzymatic acidolysis reactions, but this
strategy achieved lower production yields, and also, reaction conditions
were more aggressive: 6 hours and 60 °C (Yuksel and Sahin-Yesilcubuk,
2018). Similarly, the incorporation of lauric acid into echium TAGs was
studied to synthesize a low-calorie structured lipid (Gökçe et al.,
2013). In any case, none of these studies involved the previous
production of the oil to be used in the further enzymatic reactions.
This is a clear advantage for developing integrated processes to improve
the nutritional value of natural oils.
Conclusions
In conclusion, the present work is an original approach to develop an
integrated process to improve the nutritional characteristics of natural
oils, starting with the extraction of the raw material by
environmentally friendly methods and following with the production of
novel acylglycerols using immobilized lipases. Specifically, this work
provides relevant results for the enzymatic production of 2-MAGs under
mild reaction conditions and low temperature (25 °C) from echium seed
oil, avoiding the undesirable acyl migration. The lipase fromThermomyces lanuginosus immobilized on a hydrophobic support
reached the maximum theoretical percentage of 2‑MAGs in only 2 h.
Moreover, immobilized lipase exhibited higher stability than commercial
biocatalyst in the synthesis of 2-MAGs. The average activity after 5
cycles was 71%, allowing several reutilization cycles and developing a
feasible enzymatic process. Regarding to the fatty acid composition,
echium oil has demonstrated to be an excellent source to enzymatically
produce 2-MAGs enriched in omega‑3 SDA (up to 25%). Additionally, GC-MS
analysis demonstrated that the fatty acid composition was not modified
regarding the lipase used. Finally, the use of molecular sieves in
combination with the lipase from Rhizomucor miehei immobilized on
Sepabeads C-18 has shown to be an extraordinarily fast strategy to
produce pure STAGs in a solvent-free system (100% yield in 1h), 4 times
higher than the activity showed by commercial derivative. Hence, the
enzymatic processes developed in this study open up a range of
possibilities to synthesize acylglycerols high in stearidonic acid with
improved nutritional properties from valuable raw materials, proving the
usefulness of immobilized lipases to produce novel functional lipids.
Furthermore, the improvement of biocatalyst stability and activity by
lipase immobilization represents an important advance for its
applications at industrial levels.