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1 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Effect of lipoxygenase oxidation on surface
deposition of unsaturated fatty acids
Ali H. Tayeb1, Martin A. Hubbe1, Yanxia Zhang2 and Orlando J. Rojas1,3,*
1Department of Forest Biomaterials, North Carolina State University, Raleigh, NC 27513, USA.
2Institute of Cardiovascular Science of Soochow University, #708 Ren Ming Road, Suzhou,
215000, P.R. China
3Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University,
Espoo 00076, Finland.
ABSTRACT
We studied the interactions of lipid molecules (linoleic acid, glycerol trilinoleate and a
complex mixture of wood extractives) with hydrophilic and hydrophobic surfaces (cellulose
nanofibrils, CNF, and polyethylene terephthalate, PET, respectively). The effect of lipoxygenase
treatment to minimize the affinity of the lipids with the given surface was considered. Application
of an electroacoustic sensing technique (QCM) allowed the monitoring of the kinetics of oxidation
as well as dynamics of lipid deposition on CNF and PET. The effect of the lipoxygenase enzymes
2 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
(LOX) was elucidated with regards to their ability to reduce the formation of soiling lipid layers.
The results pointed to the fact that the rate of colloidal oxidation depended on the type of lipid
substrate. The pre-treatment of the lipids with LOX reduced substantially their affinity to the
surfaces, especially PET. Surface plasmon resonance (SPR) sensograms confirmed the effect of
oxidation in decreasing the extent of deposition on the hydrophilic CNF. QCM energy dissipation
analyses revealed the possible presence of a loosely adsorbed lipid layer on the PET surface. The
morphology of the deposits accumulated on the solids was determined by atomic force microscopy
and indicated important changes upon lipid treatment with LOX. The results highlighted the
benefit of enzyme as a bio-based treatment to reduce hydrophobic interactions, thus providing a
viable solution to the control of lipid deposition from aqueous media.
KEYWORDS: Soybean lipoxygenases; fouling; deposits; lipids; white-pine extractives;
oxidation; adsorption.
INTRODUCTION
The adsorption properties of small organic molecules such as unsaturated fatty acids (UFA)
at solid-liquid interfaces are critical in order to modify the interfacial properties in a variety of
fields, including papermaking, detergency, colloidal dispersions, wetting control, etc.1,2 Due to its
commercial importance, great attention has been paid to the kinetics of adsorption of different fatty
acids on ceramics, metal and plastic surfaces.3–5 Many water-dispersible organic components are
amphipathic, i.e., they comprise both hydrophobic and hydrophilic moieties; however, it is likely
3 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
that chemical modification of these substances, for example, by oxidation, alters their adsorption
behavior and their affinity with given surfaces. Among the different methods that have been
suggested for oxidizing UFAs,6–9 lipoxygenases (LOXs) are proposed here as alternative oxidative
enzymes. LOXs are a group of non-heme enzymes that are able to catalyze the dioxygenation of
poly-unsaturated fatty acids (PUFAs) consisting of at least one 1-cis-4 pentadiene system.10 LOXs
have been found both in plants and animal tissues, where they participate in various biological
roles.11–15 LOX main substrates include linoleic acid (C18) and arachidonic acid (C20).16–18 Among
the LOXs that have been identified,19,20 LOX-1 is easily purified/isolated and is perhaps most
relevant.21 Scheme 1 shows a simplified illustration of lipid dioxidation induced by lipoxygenases.
In reactions with equimolar amounts of linoleic acid (LA), the ferric form of the enzyme catalyzes
hydrogen abstraction from the bis-allylic (HC=CH-CH2-R) carbon in the 11th position, stereo-
specifically, which results in the formation of a pentadienyl radical complex with the ferrous
enzyme.20,22 Biomolecular oxygen (O2) reacts with the radical, to form a peroxy radical. This
unstable peroxy radical oxidizes the iron to both re-activate the enzyme and form an anion. At the
end, the hydroperoxy anion becomes protonated through the enzyme tunnel and produces the
hydro-peroxide group on the lipid chain.23–25 Even though non-enzymatic oxidation may produce
similar groups in lipids, it is unlike that enzymatic lipid oxidation leads to a racemic mixture of
hydroperoxides.26
4 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Scheme 1. Lipid dioxygenation reaction induced by lipoxygenases (R’ and R” stand for rest of the
carbon chain attaching to the lipid molecule)
There is a need to understand the interactions between lipids and polar/non-polar components,
upon LOX-induced oxidation and to identify the role of developed hydrophilic groups on their
affinity with exposed surfaces.27 Despite the interest that exists in relation to unsaturated fatty
acids, it is not yet clearly understood how the addition of a hydrophilic group on a lipid chain, via
LOX treatment, affects their adsorption properties. Here, we study the adsorption of two types of
fatty acids after LOX treatment, glycerol trilinoleate (GT) and linoleic acid (LA), as well as
isolated wood extractives. Two types of surfaces were considered as far as the extent and kinetics
of adsorption of non-polar components, namely, (1) hydrophobic polyester (polyethylene
terephthalate, PET) and (2) hydrophilic cellulose nanofibers (CNF). These materials were chosen
because of their relevance to washing, papermaking and other processes where the surfaces are
exposed to lipid colloids in aqueous media. The agglomeration of lipids in such cases may be
unwanted since it can affect negatively production or performance.28,29 This is best exemplified by
fouling (so-called deposits and stickies) of paper webs and processing fabrics, which translates
into major losses of production. Wood extractives were also considered since they are often present
5 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
in papermaking systems and owing to the fact that more than half of their composition comprises
fatty acids and triglycerides.30,31 It is conceived that the adsorption of lipid colloids on the
respective surfaces is primarily dominated by non-covalent interactions.
The dynamics of adsorption, as well as viscoelasticity of the adsorbed layer were investigated
in situ and in real time by employing a quartz crystal microbalance with dissipation (QCM-D).
The changes in lipid adsorption after oxidation were elucidated. For this purpose, a laminar-flow
stream of aqueous media containing LOX-treated lipids was passed over the QCM sensor coated
with the given film and the adsorbed mass was monitored by recording the shift in resonance
frequency.32,33 In a related context, the same technique has been used to study membrane surfaces
relevant to water treatment.34–37 Additionally, surface plasmon resonance (SPR) was used to
validate the QCM results together with atomic force microscopy (AFM), which was used to assess
the morphology of the solids upon lipid attachment. The role of solution pH and buffer was also
investigated in order to optimize the enzyme activity. This study introduces the benefits of
lipoxygenase in order to reduce hydrophobic interactions and lipid deposition. Poly-unsaturated
fatty acids (PUFAs) treated with isolated LOX are exposed to either hydrophilic or hydrophobic
interfaces, in order to minimize surface accumulation.
MATERIALS AND METHODS
The reader is referred to the notes at the end of this article for a list of abbreviations. Pure
linoleic acid, glycerol trilinoleate and soybean lipoxygenase from glycine (soybean) type I-B with
50,000 U/mg activity as well as boric acid and potassium hydroxide were obtained from Sigma
6 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Aldrich and used as received. Hexafluoroisopropanol was purchased from Fisher Scientific. All
the reagents and buffers were prepared using Milli-Q water (resistivity greater than 18 MΩ.cm).
Wood extractives were isolated from white pine wood chips using benzene-ethanol solvent
according to TAPPI standard T 204 cm-97. Polyethylene terephthalate (PET) was purchased from
Scientific Polymer NY, USA and QCM sensors from Biolin Scientific, Sweden.
LOX solutions. 10 mg of pure LOX enzymes, was added to a 20 mL liquid scintillation vial
under nitrogen gas, where the enzyme was dissolved in a 0.2 M borate and phosphate buffer
separately with constant stirring at 200 rpm. 20 mg of linoleic acid plus an equal amount of non-
ionic surfactant (Tween 20) was weighted into a 10 mL glass. Then the volume of the solution was
made up to 10 mL with distilled water. Fresh substrate was used for each test. 0.3 mL of this
solution was added to 2.7 mL of buffer with different pH, and finally 0.3 mL of LOX solution in
the buffer was added to initiate the reaction. This solution was used for the subsequent enzyme
activity tests that were determined by UV absorption, based on the formation of conjugated dienes,
the concentration of which was detected as a linear change in absorbance at 234 nm wavelength
(0.001 units per minute at 25-30 ˚C).38,39
Ultra-thin films of CNF and PET. Gold-coated QCM-D sensors were cleaned using a piranha
solution (65% v/v H2SO4 + 30% v/v H2O2 (35%)) for 10 min, and then rinsed with milli-Q water,
dried by nitrogen gas, followed by a UV/ozone cleaning for 15 min. The cleaned sensors were
used for the subsequent thin film preparation. Thin films of polyethylene terephthalate (PET) were
deposited on the sensors according to Ref.40 Briefly, a 0.15 wt% solution of PET in
hexafluoroisopropanol was prepared using PET beads that were stirred for several hours until
7 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
complete dissolution. Approximately 50-60 µL of this solution was placed on the surface of the
cleaned gold-coated sensors and spun at 3000 rpm for 20 s under an infrared lamp (250 W) that
was used to maintain the surface temperature at 85 ˚C (as monitored by an IR thermometer gun)
before and during the spinning process.
Cellulose nanofibrils (CNF) were produced by microfluidization of bleached and refined birch
kraft fibers. A sufficient amount of nanofibril gel was dispersed in milli-Q water to achieve a 1.67
g/L solids concentration. The fibrils were dispersed by using an ultrasonic micro tip homogenizer
during 10 min at 25% amplitude (Branson Ultrasonics Sonifier S-450, 400 W). The final dispersion
was centrifuged for 30 min, and the supernatant was used for spin coating. Cleaned gold sensors
were exposed to UV treatment for 15 min followed by immersion for 20 min in a 500 ppm solution
of poly (ethyleneimine) (PEI), which was used as anchoring layer. The sensors were then rinsed
with water and dried under nitrogen flow. Films were prepared by spin coating the fibril dispersion
onto the PEI-treated sensor surface at 3000 rpm for 30 s. The sensors were placed in a desiccator
until use.
Treatment of lipids and wood extractives with LOX. Two types of pure oil substrates,
glycerol trilinoleate (GT) and linoleic acid (LA), as well as white-pine extractives (WE) were used
for treatment with active lipoxygenase at room temperature. Experiments were carried out with
dispersions in a stationary air atmosphere that were subjected to magnetic stirring (200 rpm) in the
reaction vial. 10 mg of substrate was used in 10 mL of 0.2M boric buffer (pH 9.0). In order to fully
disperse the substrate, 10 minutes sonication was applied. Separately, an initial lipoxygenase stock
solution was prepared, in which 10 mg of soybean lipoxygenase was dissolved in 10 mL 0.2M
8 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
boric buffer (pH 9.0) using a magnetic stirrer. 1 mL of this solution was added to the vial containing
dispersed lipid and kept under stirring for 2 h at 200 rpm. At the end the solution was filtered for
subsequent adsorption analyses. The dispersed solution was subject to another 10 min sonication
for the purpose of evacuating the air bubbles from the solution.
Thin film characterization. AFM imaging was performed to assess the morphology,
roughness and distribution of adsorbed lipids on the thin films of CNF and PET. AFM (Nano
Scope III D3000 multimode scanning probe microscope, Digital Instruments Inc.) was used in the
non-contacting mode, in air. Scan sizes of 5×5 μm2. Three different areas on each sample were
analyzed. No image processing was employed except for flattening.
Dynamics of lipid adsorption. Adsorption and desorption phenomena on different surfaces
were followed by either a surface plasmon resonance unit (SPR, Navi 200, Oy BioNavis Ltd.,
Tampere Finland) or a quartz crystal microbalance (QCM, Model E4, Q-Sense AB, Göteborg,
Sweden). Adsorption experiments were carried out on gold-coated sensors covered with ultra-thin
films of PET and CNF under 25 C and using a laminar-flow stream of buffer solution. Milli-Q
water was used in all the experiments, and freshly prepared solutions of the oxidized fatty acids
were passed over the respective sensor. The sensors were allowed to equilibrate in the solution for
about 1 h prior to recording the base signal. Upon reaching equilibrium, signals were zeroed, and
after 10 min baseline, the buffer was replaced by lipid solution. When reaching a plateau signal in
the sensograms, lipid solution was replaced with the Milli-Q water for rinsing.
The QCM and SPR techniques were used under similar conditions (concentration, temperature,
pH, rinsing protocol, etc.) except for the flow rate, 100 vs 15 µL/min, respectively. In SPR, the
9 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
changes in refractive index of the interface was used to calculate the adsorbed layer mass and its
corresponding thickness. The change in light reflection angle due to the shift in the refractive index
of the surface correlates with any change of mass, which alter the surface plasmon, and their
associated evanescent wave. The thickness of the adsorbed lipid, d, was determined by using Eq
(1), and the adsorbed mass per unit area Γ was calculated according to Eq (2):
𝑑 =𝑙𝑑 𝛥𝜃
2𝑚(𝑛𝑎−𝑛0) (1)
𝛤 = 𝜌 . 𝑑 (2)
where Δθ is the angle change, ld is a characteristic evanescent electromagnetic field decay, ~240
nm, and m is a sensitivity factor for the sensor (109.95°/RIU) measured by calculating the slope
of Δθ for solutions of known refractive indices. no is the refractive index of the bulk solution
(buffer, 1.33) and na is the refractive index of the adsorbed species (model lipids). is the bulk
density (kg/m3).
QCM uses acoustic waves generated by an oscillating piezoelectric crystal plate to monitor
mass adsorption or desorption.41 By applying an alternating electric current to the piezoelectric
crystal quartz makes it to oscillate at the certain frequency. Adsorption or desorption of any
material on/from the crystal sensors can affect the frequency of the oscillation.42,43 The mass of
the lipid layer was determined by measuring the change in frequency (in air oscillation) of the gold
sensors before and after spin coating. The Sauerbrey equation was used as an approximation to
calculate the relative mass that is adsorbed on the give surface:41
𝛥𝑚 = −𝐶1
𝑛. 𝛥𝑓 (3)
10 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
where ∆m is the change in mass per unit area in ng/cm2 or mg/m2, ∆f is the frequency change in
Hz, C is the factor of sensitivity for the crystal (17.7 was used in this study) and n is the number
of overtone (the 3rd overtone was used for analysis). Equation (3) can be applied to determine the
mass adsorbed on the sensors provided that the adsorbed mass is smaller than the mass of the
crystal; the adsorbed layer is thin and rigid and, the adsorbed material is uniformly distributed over
the sensor surface. However, the change in sensor frequency not only depends on the adsorbed
mass but can be also affected by the viscosity and density of the adsorbed molecules.44 Therefore,
the energy dissipation is dependent on the frequency change (Δf) and the decay time (ᴦ) as follows:
𝐷 =1
𝜋𝛥𝑓𝜏 (4)
where τ values are determined by repeatedly disconnecting the oscillating crystals from the circuit
via a computer-controlled relay.
RESULTS AND DISCUSSION
Enzyme activity. Three distinct isozymes of lipoxygenase, namely, LOX-1, LOX-2 and LOX-
3 have been described to be most effective in oxidizing fatty acids.45 It is reported that different
pH optima and specific activity exist for these isozymes; while LOX-2 has the highest activity in
pH 6.5, LOX-3 is active within a larger pH range (4.5 -9.0).46 LOX-1 was used in this study for
optimizing the enzyme activity. It was prepared in acidic, neutral and alkaline colloidal solution
using two types of buffers. As shown in Figure 1b, lower enzyme activity was seen in acidic pH,
which is related to a poorer substrate solubility 47,48 and higher activity took place in alkaline
11 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
conditions. The pH of the medium can affect the polar and non-polar interactions, the conformation
of the enzyme and its active sites. Compared to PBS, LOX showed a slightly better activity in
BOR buffer. The exact reason is not well understood, but it can be due to the inhibitory effect of
the buffer components that affect protein stability.49
Figure 1. (a) UV spectrum of conjugated linoleic acid in boric buffer, representative of LOX
activity in multiple wavelength mode (b) LOX activity in different buffer at pH 5, 7 and 9
determined by UV at single wavelength (234 nm).
Surface morphology upon lipid deposition. AFM imaging revealed lipid agglomeration on
the tested surfaces and further indicated the role of substrate morphology on the lipid-lipid and
lipid-substrate interactions. As can be observed in images of adsorbed white pine extractives (WE)
(Figure 2, a1’-a2’), aggregates were mostly formed on PET. This observation agrees with the
results obtained from QCM/SPR experiments to be discussed next and provides evidence of the
lipid adsorption on polyester. The sizes of the lipid clusters were notably reduced when lipids were
12 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
treated (oxidized) with LOX, particularly when PET was used as substrate. The PET surfaces had
a higher roughness compared to those of CNF, and, at least to some limited extent, adsorption of
the white pine extractives increased the roughness (Table 1). This may suggest that the
agglomeration of hydrophobic components mostly took place on the upper layers of the film rather
than areas underneath the surface. However, this phenomenon was less significant in the case of
LOX-treated WE, given the fact that the oxidized lipids showed less affinity to polyester and the
change in the film roughness was less notable. It was observed that the surface roughness of the
CNF films followed similar behavior as that for PET; the deposition of lipids caused a slight
increase in roughness. However, less agglomeration was observed on the cellulosic film compared
to PET (Figure 2, b1’, b2’).
Table 1. Roughness values of PET and CNF thin films for AFM height images.
PET/CNF Film Roughness (nm)
a-(PET) 65.5
a1-(PET-WE) 70.6
a2-(PET-WE-LOX) 68.1
b-(CNF) 6.10
b1-(CNF-WE) 10.8
b2-(CNF-WE-LOX) 8.10
13 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Figure 2. Non-contact mode AFM images at 5×5 µm2 for PET and CNF films, a) PET, b) CNF.
a1 and b1 correspond to the surfaces after adsorption of unmodified WE. a2 and b2 correspond to
adsorption of enzymatically treated WE. The panels indicated as a1’, b1’, a2’, b2’ are the
corresponding phase images. The height and phase scale corresponds to z values as indicated by
the height bar.
14 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Lipid affinity with PET and CNF. Generally, lipid components have a surface tension close
to those of adhesives (~32 mJ/m2),50 and their affinity with surfaces tends to increase when the
surface free energies are similar. This explains why wood extractives have a tendency to deposit
onto hydrophobic surfaces. Using the condition that indicated the best LOX activity (pH,
temperature and buffer type), similar systems were applied in heterogeneous phase, i.e., lipid
adsorption on solid surfaces after enzyme treatment. The dynamics of adsorption of the model
lipids as well as wood extractives are illustrated in Figure 3a and 3b for PET (hydrophobic) and
CNF (hydrophilic) surfaces, respectively, under the same conditions. The negative shift in
resonance frequency is associated with lipid adsorption on surfaces. The adsorption of the pure
lipids and wood extractives decrease upon oxidative (LOX) treatment. Rinsing with buffer at time
= 50 min was performed and continued until time = 60 min. Irreversible mass adsorption was
observed in all cases, as indicated by the fact that the frequency recorded after rinsing did not
return to the respective baseline. A faster adsorption process was observed on the PET surfaces,
especially in the case of WE (Figure 3a). The QCM data show the irreversible adsorption of
lipophilic components on both surfaces even after the enzymatic treatment, but, overall, the
adsorption onto polyester-based films (PET) was higher and faster compared to that on the
hydrophilic cellulose. The adsorption rate was significantly reduced following the oxidation
process. In the case of CNF, the changes in the lipid affinity as a result of oxidation were more
limited, especially for wood extractives adsorption. Interpretation of the results in the case of the
cellulosic surfaces is more complex since they respond to the environment and are soft. Also, water
coupling or hydration (changes in swelling and density) are expected.
15 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Figure 3. Representative sensograms showing the shift of QCM frequency (3rd overtone) as a
function of time upon adsorption of unmodified lipids; WE, LA and GT, after LOX-treated lipids;
WE-LOX, LA-LOX, and GT-LOX. The adsorption occurs in aqueous media (1mg/mL, pH 9.0,
16 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
25 ˚C) under constant flowrate of 100 µL/min. The surfaces correspond to (a,c) PET and (b,d)
CNF.
SPR analysis. The adsorption isotherms for the pure lipids and WE on PET and CNF
surfaces, before and after lipoxygenase treatment were also acquired by SPR techniques, and
results are summarized in Table 2. The SPR was used to validate the QCM results. By comparing
the results of acoustic (QCM) and optical (SPR) methods, it was possible to monitor the changes
in the film mass and thickness51 and to follow the kinetics of adsorption, lipids coverage and
molecular conformation.52,53 Compared to SPR, the QCM mass was overestimated for lipid
adsorption, given the contribution of water coupling; however, the trends in the change of adsorbed
mass onto CNF and PET calculated with both techniques were similar. It is apparent that pre-
treatment of lipids with the enzyme under an optimized condition (boric buffer, pH 9.0, 25 ˚C)
reduce lipid deposition on PET surfaces.
Table 2. Lipids mass adsorption (mg/m2) from an aqueous media measured by QCM and SPR on
thin films.
Adsorbed
mass
(mg/m2)
Lipid
PET CNF
Before-LOX After-LOX Before-LOX After-LOX
ΓSPR
LA 1.8 1 1.4 1.3
GT 2.0 1 4 2.6
WE 6.3 2.1 2.2 1.7
ΓQCM
LA 8.9 3.3 7.6 5.2
GT 11.5 4.3 8.8 3.8
WE 34.1 11.8 6.9 4
17 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
Lipid layer dissipation. QCM ΔD-Δf curves (dissipation vs frequency profiles) for lipid
adsorption on CNF and PET are included in Figure 4 a, b. The analysis of energy dissipation
provides additional information about the lipid behavior with or without oxidation. The
measurement of changes in dissipation relates to the viscoelastic properties of the lipid layer. In
general, compact and rigid adsorbed layers lead to relatively smaller changes in dissipation
compared to that of a loosely attached layer. According to the results, for PET substrates, the
energy dissipation for unmodified WE and GT are lower than LA. The large dissipation values for
the untreated LA suggest a more extended attachment of the adsorbed lipid, and this behavior tends
to continue even after the lipid was treated with LOX. However, the increase in slope of the
dissipation profiles for GT and WE after LOX treatment (Figure 4a) was more noticeable upon
adsorption on PET, indicating a different adsorption kinetics. This implies the more complex
components (GT and WE) make a less rigid contact on PET after the oxidation, which probably is
due to some repulsion between the lipid molecules and the PET surface. Furthermore, it seems the
layers formed as a result of oxidized GT and WE were more viscoelastic compared to their
corresponding unmodified lipids, which were more rigid.
The loss of energy dissipation for the oxidized lipids imply a loose attachment of the
components to PET and can be due to the formation of new lipid structure as a result of enzyme
oxidation: the addition of polar groups on the lipid chain may cause the lipid to be slightly more
hydrophilic. This trend however was not seen for LA, which showed almost a linear relation in the
profiles. Apparently, the configuration of the modified LA cause the same relative dissipation (a
18 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
lower adsorbed mass and energy dissipation are recorded). From the same figure, one can conclude
that oxidized GT and WE adsorb on PET as a more extended, viscoelastic layer.
Figure 4. Changes in dissipation vs frequency upon adsorption of lipids (1mg/mL, pH 9.0) with
(LA-LOX, GT-LOX, WE-LOX) and without (LA, GT, WE) LOX enzymatic treatment on (a) PET
and (b) CNF.
Unmodified wood extractives adsorbed on PET to a large extent but caused little change in
dissipation. Thus, a rigid layer was formed. However, a slight increase in the dissipation for lipids
that were treated with the enzyme was observed, indicating that the oxidized components formed
a looser adsorbed layer.
Tests with hydrophilic cellulose indicate that compared to the untreated lipids, most of the
oxidized lipophilic components exhibited lower dissipation (Figure 4b). This is in agreement with
the fact that the addition of polar groups to the fatty acids make them less hydrophobic and, as a
19 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
result, the lipid adsorbed on CNF becomes more densely packed. Besides, Figure 4b, d, shows a
difference in lipid adsorption on cellulose after enzymatic treatment, with a lower dissipation.
Linoleic acid, which is a linear and small fatty acid, showed a different behavior, and the loss of
dissipation was more significant for this fatty acid upon LOX oxidation. Probably, the modified
LA, which is more hydrophilic, engages in hydrogen bonding with the primary alcohols on CNF
and, as a result, the lipid adsorbs more densely. However, the dissipation decreased for WE and
GT on CNF almost linearly with the amount adsorbed on the surface. The addition of polar groups
to the lipid may increase hydration and increase the likelihood for removal of the lipid during
washing or rinsing steps.
CONCLUSIONS
Experiments with electroacoustic and optical methods (QCM and SPR) suggested that lipids
that were enzymatically modified with lipoxygenases (LOX) had a lower affinity to both lipophilic
polyester and hydrophilic cellulose, owing to the changes in surface free energy. The alteration in
the dissipation suggested a change in the hydration and viscoelasticity of the lipids that adsorbed
on the surfaces. AFM images indicated limited lipid aggregation as well as changes in PET surface
roughness upon enzymatic (LOX) treatment. The treatment with LOX of lipid colloidally
suspended in aqueous matrices inhibited their deposition of solid surfaces, signifying an
environmentally-safe method to control fouling in related processes.
20 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
AUTHOR INFORMATION
Corresponding Authors
*Mailing address: Department of Bioproducts and Biosystems, School of Chemical Engineering,
Aalto University, FI-00076, Espoo, Finland. E-mail: [email protected]
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
Abbreviations used in this paper.
Abbreviation Description
PET poly(ethylene terephthalate)
CNF cellulose nanofibrils
PEI poly (ethylenimine)
QCM quartz crystal microbalance
SPR surface plasmon resonance
LOX lipoxygenase
BOR boric buffer
PHS phosphate buffer
WE white pine extractive
WE-LOX extractives treated by lipoxygenase
LA linoleic acid
LA-LOX linoleic acid treated by lipoxygenase
GT glycerol trilinoleate
GT-LOX glycerol trilinoleate treated by lipoxygenase
21 Author version. Citation: Langmuir 33(18), 4559-4566; DOI: 10.1021/acs.langmuir.7b00908
ACKNOWLEDGMENTS
The authors acknowledge support from United Soybean Board (USB) under Project Number
1640-512-5276 and HYBER’s Academy of Finland’s Centers of Excellence Program (2014-
2019). We thank Dr. Keith D. Wing for fruitful discussions and advice.
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Table of Contents Graphics (TOC)
Effect of lipoxygenase oxidation on surface deposition of unsaturated fatty acids
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Lipid adsorption on PET
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Lipid adsorption on PET
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Change in lipids viscoelasticity