Food Chemistry 439 (2024) 138149

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

“Characterization and stability of α-tocopherol loaded solid lipid

nanoparticles formulated with different fully hydrogenated vegetable oils”
Marcella Aparecida Stahl a, *, Fernanda Luisa Lüdtke a, b, c, Renato Grimaldi a,
Mirna Lúcia Gigante a, Ana Paula Badan Ribeiro a
a
Department of Food Engineering and Technology, Faculty of Food Engineering, University of Campinas, 13083-862 Campinas, Brazil
b
CEB - Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal
c
LABBELS – Associate Laboratory, Braga/Guimarães, Portugal

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

Keywords: Solid lipid nanoparticles can be compatible with several bioactive compounds and confer a differentiated
Lipid nanoparticles crystalline structure. This study aimed to produce α-tocopherol loaded solid lipid nanoparticles with fully hy­
vitamin E drogenated oils and fats from palm oil, soybean oil, and crambe oil, by high-pressure homogenization, using
Hardfats
lecithin as an emulsifier. After recrystallization of solid lipid nanoparticles, dispersions were evaluated until 60
Crystallinity
days of storage for particle size, polydispersity index, zeta potential, microstructure, dispersion stability and
α-tocopherol quantification. α-tocopherol loaded solid lipid nanoparticles showed particle sizes and zeta po­
tential values considered adequate for this type of particle. Presence of α-tocopherol altered thermal behavior of
the particles, leading to increased crystallinity, with no changes in polymorphism, when compared to the
unloaded solid lipid nanoparticles. All α-tocopherol loaded solid lipid nanoparticles dispersions showed stability
with no losses of α-tocopherol, indicating their potential as a carrier for this compound in fortified foods.

1. Introduction protected and released from the food matrix for absorption in the
gastrointestinal tract (McClements et al., 2015). Studies have shown the
Lipophilic compounds are involved in metabolic processes, and due effectiveness of nanoemulsions for encapsulation of vitamin E and its
to their low availability caused by some dysfunction in the organisms, derivatives in edible emulsions delivery systems and drug (Yang and
dose needed by the body can be guaranteed by food supplements and McClements, 2013; Oliveira et al., 2016; Oehlke et al., 2017, Teixeira
nutraceuticals. Typically, lipophilic compounds are classified as having et al., 2017), mainly due the capacity of these systems to improve the
low solubility in aqueous systems and variable permeability, thus chemical stability of the vitamin, potential bioavailability, and antiox­
identification of the best system for encapsulation of these components idant properties in pharmaceutical and food products (Teixeira et al.,
is a major challenge (Dima et al., 2015). Vitamin E is a fat-soluble 2017).
vitamin naturally synthesized by photosynthetic organisms, consisting Composed by lipid matrices with high melting point, solid lipid
of a group of chemical compounds formed by tocopherols and toco­ nanoparticles (SLN) are one of the most promising lipid delivery sys­
trienols. These tocopherols are found in eight different forms, and tems. Due to the larger surface area, SLN are expected to be biologically
α-tocopherol form is the most active in humans, considered one potent more active than their pure chemical composition, with prospects as
biological antioxidant, with 100 % vitamin E activity. It acts in pro­ functional foods with bioactive compounds, improving bioavailability of
tecting cells against the effects of free radicals by stopping the propa­ poorly water-soluble substances (Shah et al., 2014; Mohamed and
gation of lipid oxidation (Feng et al., 2009; Pacífico et al., 2012; Ziani Mohamed, 2019). The type of solid lipid and emulsifier used to produce
et al., 2012). Optimal intake comprises the daily intake necessary to SLN has been shown to play a pivotal role in the characteristics and the
exert biological functions and for health promotion and disease pre­ stability of the particles (Zafeiri et al., 2017), and also in the entrapment
vention (Galli et al., 2017). efficiency (EE) of the bioactive compound. Lipids used to produce SLN
To exert its biological functions, however, α-tocopherol must be must be solid at room and body temperature, biocompatible and

* Corresponding author.
E-mail address: m145046@dac.unicamp.br (M. Aparecida Stahl).

https://doi.org/10.1016/j.foodchem.2023.138149
Received 1 February 2023; Received in revised form 31 July 2023; Accepted 3 December 2023
Available online 6 December 2023
0308-8146/© 2023 Elsevier Ltd. All rights reserved.
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

biodegradable, well tolerated by the physiological system, and generally were 700 bar and two homogenization cycles (Ludtke et al., 2022; Stahl
recognized as safe (GRAS) (Shah et al., 2014). In addition to lipids and et al., 2023). At last, nanoemulsions were cooled at 25 ◦ C for 24 h to
water, emulsifiers are also part of the SLN composition, with the main recrystallize lipid phase and obtain dispersions containing SLNα, which
function of stabilizing particles and ensuring higher performance (Klang were stored at the same temperature for stability study (Qian et al.,
and Valenta, 2011). 2013; Kumbhar and Pokharkar, 2013; Yang et al., 2014).
Lecithin is the most widely used natural emulsifier in the food in­
dustry. It is among the safest and most biocompatible emulsifiers with
GRAS regulatory status and is considered safe for use in nanoparticles 2.3. Characterization of SLNα
even in its modified forms (Klang and Valenta, 2011). The most
commonly used lipid matrices to obtain tocopherol loaded SLN are pu­ SLNα structures were evaluated for particle size (PS), polydispersity
rified and synthetic materials, like glyceryl tristearate (Oehlke et al., index (SPAN), Zeta potential (ZP), morphology by polarized light mi­
2017) and glyceryl behenate (de Carvalho et al., 2013). In this sense, croscopy (PLM), Turbiscan stability index (TSI), solid fat content (SFC),
fully hydrogenated oils commonly referred as hardfat, like a soybean thermal behavior by differential scanning calorimeter (DSC), and poly­
(Ludtke et al., 2023; Stahl et al., 2023), palm and crambe (Silva et al., morphism by X-ray diffraction. The stability of α-tocopherol in the
2022; Stahl et al., 2023) can be used as an alternative to synthetic lipids nanodispersions over time were also evaluated. These properties were
to successfully produce lipid nanoparticles to entrap and deliver bioac­ evaluated as a function of fatty acid chain length and effect of
tive compounds. Obtained by the total hydrogenation of oils and fats, a α-tocopherol stability on days 1, 7, 15, 30, and 60 after obtaining and
lipid modification process in which all carbon–carbon double bonds are recrystallizing lipid nanostructures at 25 ◦ C in an incubation chamber
saturated, hardfats are formed by high melting point triacyclglycerols (Nik, Langmaid, and Wright, 2012; Das, Ng, and Tan, 2012; Kumbhar
(TAGs) (Ribeiro et al., 2013). To date, few studies have used fully and Pokharkar, 2013).
saturated fatty acids from conventional oil sources for SLN production.
Therefore, this study aimed to produce and characterize α-tocoph­ 2.3.1. Particle size (PS), polydispersity index (SPAN)
erol loaded SLN-type structures formulated from different hardfats. In distribution, PS, and SPAN of SLN were determined by laser
this study, hardfats obtained from sources of conventional vegetable oils diffractometer using a Mastersizer 2000 (Malvern Instruments). For
were used to produce SLN using lipid matrices with low cost of pro­ that, SLN aliquots were mixed with distilled water under stirring (1750
duction and widely used in food. For that, palm, soybean, and crambe rpm) until reaching an obscuration rate of 2 %. The oil refractive index
hardfats have been chosen to evaluate the effect of different sizes of fatty and particle absorbance values used in Malvern software were 1.45 and
acid chains in the SLN characteristics and in the stability of α-tocopherol 1.33, respectively. The analyses were performed in triplicate at room
into these nanostructures. As far as we know, there is no scientific study temperature (Averina et al., 2011). The mean PS values of the SLN were
which evaluates the α-tocopherol stability in SLN produced with expressed in terms of the mean surface diameter (d3,2) obtained using
hardfats. equation (1), and the polydispersity was determined by calculating the
SPAN (Equation (2)).
2. Material and methods ∑
nidi3
d3,2 = ∑ (1)
nidi2
2.1. Material
d90 − d10
All hardfats were obtained by total hydrogenation process. Fully SPAN = (2)
d50
hydrogenated palm oil (FHPO) was supplied by Agropalma (Brazil),
fully hydrogenated soybean oil (FHSO) was supplied by SGS Group where ni is the number of particles with diameter di and d10, d50, and
(Brazil), and fully hydrogenated crambe oil (FHCO) was supplied by d90 represent 10 %, 50 %, and 90 % of the cumulative droplet volume,
Cargill Foods (Brazil). The major fatty acid composition of these hardfats respectively.
was: FHPO (C16:0 - 38.83 %; C18:0 - 57.90 %), FHSO (C16:0 - 10.70 %;
C18:0 87.31 %), and FHCO (C18: 0 31.07 %; C22:0 - 57.33 %), where 2.3.2. Zeta Potential (ZP)
C16:0, C18:0, and C22:0 correspond to palmitic (P), stearic (S), and ZP was determined using the Zetasizer Nano-ZS instrument (Malvern
behenic (Be) acids, respectively. Concerning TAGs composition, pre­ Instruments, Malvern, UK). Samples were diluted (1:100) in distilled
dominant trisaturated fatty acids were FHPO (PPS − 38.81 %; PSS − water for later reading on the equipment (Averina et al., 2011). The
40.55 %; SSS − 12.20 %), FHSO (PSS − 33.71 %; SSS − 62.67 %), FHCO analyses were performed in triplicate at room temperature.
(90.86 % of TAGs with Be, especially SSBe, SBeBe, BeBeBe). Enzymat­
ically modified lecithin (SOLEC AE IP) with 16 % lysophosphati­ 2.3.3. Solid fat content (SFC)
dylcholine (LPC) and 26 % phosphatidylcholine (PC) supplied by Solae SFC was determined using Bruker pc120 Minispec low-resolution
(Brazil) (HLB 7–8) was used as an emulsifying agent. Lipophilic bioac­ pulsed Nuclear Magnetic Resonance (NMR) Spectrometer with aid of
tive compound DL-all-rac-α-tocopherol with 96 % purity produced by high precision dry bath (0–70 ◦ C) Tcon 2000 (Duratech, USA). Samples
Sigma-Aldrich (USA) was used. were transferred to NMR tubes after recrystallization of SLNα and stored
in the same condition of stability (25 ◦ C) for subsequent measurement of
2.2. Production of SLN solid fat contents (Nik et al., 2012).

SLN dispersions were made with 5 % (w/w) hardfat (FHPO, FHSO, or 2.3.4. Thermal melting behavior
FHCO), and 1 % α-tocopherol based on the lipid fraction, which Thermal analysis was performed on a TA Q2000 differential scan­
comprised lipid phase. Aqueous phase (95 %) consisted of 1 % (w/w) ning calorimeter (DSC) coupled to RCS90 Refrigerated Cooling System
lecithin in ultrapure water as an emulsifier. To prevent microbial growth (TA Instruments, Waters LLC, New Castle). Data were analyzed using
in the emulsions, 0.02 % sodium azide was added to the aqueous phase. Universal V4.7A processing system (TA Instruments, Waters LLC, New
Lipid and aqueous phases were heated to 85 ◦ C and stirred using a T18 Castle). Analysis conditions were: sample mass: ~ 10 mg; a 25 ◦ C
ultra-turrax (IKA, Germany) for 3 min at 15,000 rpm. Aqueous disper­ isotherm for 10 min; melting events from 25 to 100 ◦ C, at 10 ◦ C/min
sions of SLN containing α-tocopherol (SLNα) were produced using hot (Wang et al., 2014). SLNα dispersions were weighed in airtight
HPH technique in a high-pressure homogenizer Homolab 2.20, Buffalo aluminum pans (TA Instruments, Waters LLC, New Castle) after
series from FBF (Italy). The process conditions used to produce the SLN recrystallization time, and all measurements were performed in the

2
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

same small pan, stored at 25 ◦ C in an incubation chamber. Following 3. Results and discussion
parameters were analyzed: onset melting temperature (Tom), peak
melting temperature (Tp), melting enthalpy (ΔH), and final melting 3.1. Particle Size, polydispersity index and Zeta potential
temperature (Tfm).
PS is an important characteristic of nanoparticles that can be affected
2.3.5. Polymorphism by the experimental conditions including SLN composition, formulation
Polymorphic form of SLNα was determined by X-ray diffraction, variables such as emulsifiers and incorporated compounds, structure
according to the AOCS Cj 2–95 method (AOCS, 2009). Analyses were and properties of lipids and compounds, and processing conditions (Saez
performed in a Philips diffractometer (PW 1710), using Bragg-Bretano et al., 2018). In addition, PS is also related to the release process, and the
(θ: 2θ) geometry with Cu-K radiation (λ = 1.54056 Å, 40 KV, and 30 larger nanoparticles, the slower their dissociation (Katouzian and Jafari,
mA). Measurements were obtained with steps of 0.02◦ in 2 θ and an 2016).
acquisition time of 2 s, with scans from 5 to 40◦ (scale 2 θ). SLNα dis­ Fig. 1a shows the PS, expressed as the mean diameter (d32) of SLNα,
persions were analyzed at 25 ◦ C. Polymorphic forms were identified obtained with different hardfats. PS range was from 164 to 188 nm. No
from the characteristic short spacing of the crystals. Contents of different significant difference (P < 0.05) was observed for PS between samples
types of crystals were estimated by the relative intensity of short spacing on day 1 of storage. However, samples showed variations in PS over
(AOCS, 2009). All results were expressed as mean and standard devia­ time, as shown in Fig. 1. After 60 days, all SLNα showed increased (P <
tion of triplicate determinations for all parameters. 0.05) PS when compared to day 1 of storage. The increase of PS was
more pronounced in SLNα produced with FHCO.
2.3.6. Morphology PS of the SLNα increased during 60 days of storage. Increase in PS of
Morphology of the SLNα dispersions was analyzed by polarized light SLNα may be due to the presence of bioactive compounds (Saez, 2018),
microscopy (PLM) using an Olympus model BX 50 polarized light mi­ although some authors reported that compound may not affect PS
croscope (San Jose, USA) coupled with a digital camera (Media Cyber­ (Oehlke et al., 2017). In this study, PS were lower than 500 nm, which is
netics, Bethesda, USA). SLNα dispersions were placed on microscope desired for possible food applications (Dan, 2016).
slides after 24 h of preparation and analyzed throughout stabilization As observed for PS, SPAN values of the SLN indicated the degree of
times. Slides were placed on the plate support (Mettler Toledo, FP82 stability and quality of the PS distribution (Fig. 1b). Samples with SPAN
Microscope Hot Stage, Columbus, USA), with image acquisition at 25 ◦ C, values greater than 1 indicate a wide distribution and high poly­
corresponding to stabilization temperature, at 40x magnifications dispersity (Songsurang et al., 2011; de Morais Ribeiro et al., 2018). SLNα
(Kovacevic et al., 2014). Images were captured and the clusters were showed slightly higher values when compared to the control, which
identified by software Image-Pro Plus version 7.01 (Media Cybernetic, maintained or decreased over time. Dispersions SLNα FHPO and SLNα
Bethesda, EUA). FHSO showed stability over time. SLNα FHCO maintained its stability in
the highest SPAN until day 30 of storage when compared to the other
2.3.7. Physical stability dispersions, with a reduction on day 60, with an opposite tendency to
Physical stability of SLNα dispersions was evaluated in a Turbis­ that observed for PS. Dispersion SLNα FHCO showed a significant
canLab® optical analyzer (Formulaction, France), and data were decrease in SPAN at day 60 along with an increase in PS, indicating
analyzed by TurbiSoft 2.0 software. For that, SLNα dispersions were lower polydispersity. These results may affect stability and appearance
placed in a cylindrical glass cell at 25 ◦ C. Detection was performed by an of the final product. FDA has established no acceptable limit for poly­
infrared light source with a length of 880 nm, with transmission (T) and dispersity index values, thus specific standards should be established by
backscattering (BS) detectors. T detection measures the light that passes the responsible regulatory agency for different applications (Danaei
through the sample, while BS receives backlight not absorbed by the et al., 2018).
sample. TSI corresponds to a cumulative sum of BS or T and is associated Changes in SPAN showed corresponding variation of PS and SLNα
with overall destabilization of the sample in a given time. This param­ polydispersity, represented by values above 1.
eter provides information about the possible destabilization mecha­ ZP results are a measure of the surface charge. Normally, the values
nisms, which can be reversible upon particle migration (cremation or above |30 mV| indicate more repulsion of particles and suggesting that
sedimentation) or irreversible, with changes in particle size (flocculation SLNα were electrostatically stable (Carvalho et al., 2013). All SLNα
or coalescence) affecting overall stability of the sample (Silva et al., dispersions showed ZP values above the recommended, as shown in
2011; Formulaction, 2021). Fig. 1c. Although a tendency of decreasing ZP was observed from day 1
to day 60, high values were observed throughout the period studied.
2.3.8. α-tocopherol stability ZP values of SLNα were close to those of the control SLN (Stahl et al.,
For the analysis, SLNα were frozen at − 40 ◦ C in an ultra-freezer 2023) and also decreased over time, although all values were above |30
(model UK 05 Klimaquip, Brazil), freeze-dried in a freeze-dryer (model mV|, indicating stable dispersed systems. de Carvalho et al. (2013)
L108, Liotop, Brazil), and homogenized at the time of analysis. studied SLN with and without addition of α-tocopherol and reported a
α-Tocopherol content was determined in the dispersions using UV decrease in ZP over time for samples stored at 20 ◦ C, with higher values
UV–Vis. Spectrophotometer Orion AquaMate 8000 (Thermo Fisher Sci­ when compared to samples without bioactive compound, which indi­
entific, Waltham, Massachusetts, USA) at 290 nm, using calibration cated that α-tocopherol avoided agglomeration between the particles
curve FI = 8454.5 x [vitamin], R2 = 0.9998, diluted in analytical grade and consequently gelation of the dispersion. Teixeira et al. (2017)
tetrahydrofuran (THF). evaluated SLN with different and similar α-tocopherol concentrations
and reported ZP values close to the control. These findings indicate that
2.3.9. Statistical analysis ZP values are more susceptible to changes as a function of the lipid used
All analytical determinations were performed in triplicate. The re­ rather than presence of α-tocopherol.
sults were analyzed by Analysis of Variance (ANOVA) and Tukey’s test
for comparison of means at a 5 % significance level using STATISTICA 3.2. Solid fat content
7.0 software (Stat-Soft Inc, Tulsa, OK, USA).
The determination of SFC is a good indicator of the degree of crys­
tallinity presented by lipid nanoparticles, since it is related to the crys­
tal’s formation, the presence of crystalline agglomerates, and the crystal
lattice (Ludtke et al., 2022). Crystallization behavior of SLN over time

3
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

Fig. 1. Effect of the hardfats on (a) Particle size (nm), (b) Polydispersity index and (c) Zeta Potential of SLN lecithin-based with alpha-tocopherol (SLNα). FHPO: fully
hydrogenated palm oil; FHSO: fully hydrogenated soybean oil; FHCO: fully hydrogenated crambe oil, d: days. Errors bars represent the standard deviation of n = 3
replicates. A-C Different capital letters indicate significant difference (P < 0.05) between SLNα obtained with different hardfats on the same storage day. a-b Different
lower-case letters indicate significant difference (P < 0.05) for the same SLNα over the storage period (1 to 60d).

was mainly affected by various factors, including composition of lipids 3.3. Thermal melting behavior
used and their interaction with emulsifiers, production process, and
presence of bioactive compounds. Melting behavior was analyzed by heat flow as a function of tem­
Fig. 2 shows the SFC, at 25 ◦ C, of SLNα obtained from FHCO, FHPO perature variation (by DSC). SLN with different hardfats were compared
and FHSO. Regarding SFC of SLNα at 25 ◦ C, dispersion SLNα FHPO with and without the bioactive compound, aimed at monitoring possible
showed stability throughout the time, with no significant differences degradation. Melting behavior of TAGs can be modified to a greater or
between the results, with 6.7 % on day 60 of storage. In turn, SLNα FHSO lesser degree with addition of emulsifiers and encapsulated compounds
and SLNα FHCO showed a mild decline, with SFC of 7.6 and 6.4 %, and (Oehlke et al., 2017; ur Rahman et al., 2020).
7.7 and 6.8 % on days 1 and 60, respectively, needing further evaluation The parameters used to discuss the thermal melting behavior for the
to conclude whether there was a significant trend. SLNα obtained from FHCO, FHPO and FHSO are found in Fig. 3 and
SFC of the SLNα determined by NMR at 25 ◦ C showed a higher Table SM 1 and correspond to the onset melting temperature (Tmo),
percentage of solid fat when compared to the control (Stahl et al., 2023), which refers to the beginning of the solid–liquid transition; final melting
with a decline over time. This difference was not relevant for the par­ temperature (Tfm), which corresponds to the temperature at which the
ticle, since it did not affect parameters such as particle size or enthalpy thermal melting effect was completed; melting peak temperature (Tp),
values in the thermal behavior, which represent changes in crystallinity which indicates the point at which the maximum thermal effect
of the particle. However, this variation in the SFC can be related to the occurred; and, melting enthalpy (J/g), which reflects the energy
variation in stability of the SLN dispersions, indicated by the TSI value, required for the phase change to occur.
which will be discussed next. Dispersion SLNα FHPO showed lower SFC, Dipersions of SLNα exhibited a single melting event for all periods
with no significant difference over time, and was the most stable studied. Dispersion SLNα FHPO, which was composed of hardfat with
dispersion according to TSI and higher ZP values. Dispersions SLNα small-chain TAGs, showed the lowest temperatures and a subtle increase
FHSO and SLNα FHCO presented higher results of the SFC on day 1. in enthalpy on day 60 when compared to day 1. In turn, SLNα FHSO and
After 15 days, these values showed significant decreases, accompanied SLNα FHCO showed no change in crystallinity as a function of bioactive
by the loss of stability. compounds, as shown in the enthalpy constant values.
SLNα exhibited changes in the melting parameters when compared

4
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

Fig. 2. Solid Fat Content (%) of SLNα at 25 ◦ C. FHPO: fully hydrogenated palm oil; FHSO: fully hydrogenated soybean oil; FHCO: fully hydrogenated crambe oil;
d: day.

Fig. 3. Melting events of control SLN (1d) and SLNα obtained with a) fully hydrogenated crambe oil, b) fully hydrogenated palm oil and c) fully hydrogenated
soybean oil over the storage time (1 to 60d).

to unloaded SLN (Stahl et al., 2023), regardless of the hardfat used. SLNα a disordered crystal matrix, with a consequent reduction in crystallinity,
showed decrease in Tom, Tp, and Tfm, and increase in enthalpy from which can provide more space for accommodation of compound mole­
day 1 of storage, indicating changes in crystallinity with the presence of cules. Consequently, there is a greater enthalpy variation once crystal­
α-tocopherol. Carvalho et al. (2013) studied optimization of α-tocoph­ line structure is not fully stable, requiring more energy to melt
erol incorporation into the SLN and reported similar results in this study, crystalline portions. Oehlke et al. (2017) reported that modifications
with lower Tom for SLNα when compared to the corresponding unloa­ depend on the α-tocopherol concentration, and this compound is prob­
ded SLN. Interaction of α-tocopherol with crystals of lipid matrix creates ably located in the lipid matrix, at the interface with the emulsifier

5
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

rather than the core, directly affecting melting behavior and structure forms were also found for SLNα FHSO and SLNα FHCO, dispersion SLNα
rearrangements. Furthermore, the liquid state of α-tocopherol at room FHSO exhibited a low intensity of the peak (Table 1) corresponding to
temperature may also have led to a decrease in melting temperature the presence of β’ polymorph, represented by short spacing at 4.2,
when compared to unloaded SLN (Stahl et al., 2023). Although results indicating possible onset of β-stabilization.
no showed significant differences, thermograms exhibited a slight Use of emulsifiers that act on the crystallization process, such as
variation of the peak over time, evidenced in 60 days. This alteration in lecithin, may help control crystal growth. In this study, the presence of
the peak can be related to changes in polymorphic transitions, that affect α-tocopherol did not affect polymorphism of lipids when compared to
α-tocopherol release or activity after prolonged study times (Salminen unloaded SLN (Stahl et al., 2023). Shukat et al. (2012) reported that
et al., 2016; Oehlke et al., 2017). It is important emphasize that the decrease in PS and SFC was accompanied by increase in β-poly­
samples remained stabilizing in the same pan. This process simulates the morphism, observed for all samples and all periods studied. Salminen
recrystallization of the particles as the samples melting and then et al. (2016) reported no impact of α-tocopherol on the crystallization
recrystallized during storage, cooperate with studies, and elucidate the behavior and suggested that mechanism of SLN with α-tocopherol leads
changes shown in thermograms (Fig. 3). to an accumulation of bioactive compound on particle surface, as
moderate hydrophobicity can cause compound to diffuse into the
3.4. Polymorphism aqueous phase. Migration into the lipid phase is limited upon cooling
due to the onset of crystallization, leading to a rapid release of the
Knowledge of crystallization behavior of a solid matrix is important compound, as well as making it susceptible to oxidation. Oehlke et al.
for protection against instability and release control of encapsulated (2017) reported that SLN incorporated with α-tocopherol did not affect
compounds (Bahari and Hamishehkar, 2016). Crystallization during polymorphic habit of tristearin, showing similar behavior to pure SLN.
storage can lead to alteration of polymorphic forms, thus changing However, results of melting behavior of this study indicated changes in
stability, conformation, and packing of the molecules, which can result the crystal structure that contributed to entrapment of the lipophilic
in release of the bioactive compound. Sustained distribution is often compound, despite a single peak representing polymorphic stability, as
related to metastable β’ polymorphism. In addition, transition to the observed for respective controls. Furthermore, the evaluation over 60
more stable polymorphism (β polymorph) leads to a more ordered days showed that there was no fusion or recrystallization of SLN to less
rearrangement, which can also lead to release of incorporated com­ stable polymorphic forms, which is very positive when considering the
pounds (Müller et al., 2000; Heurtault et al., 2003). application of these nanostructures in foods.
Polymorphism of SLNα analyzed by X-ray diffraction showed peaks
referring to polymorphic forms, with some interference due to the water 3.5. Morphology
present in the emulsion, which did not affect identification. Short
spacings, peak intensity, and identification of polymorphic forms found Morphology of the SLNα dispersions is shown in Figure SM 2. Sam­
for SLNα are described in Table 1. Both β’ and β forms were observed for ples exhibited polydispersity, with presence of clusters and homoge­
SLNα FHPO for all periods studied. Although the same polymorphic neous dispersion. Concerning the number of clusters, dispersion SLNα
FHPO showed 56 to 58 % clusters over time, while SLNα FHSO ranged
from 60 to 64 %, and SLNα FHCO showed 51 to 59 % clusters.
Table 1
Short spacing and polymorphic forms of lecithin-based SLNα, at 25 ◦ C*. PLM showed that SLNα dispersions presented homogeneous
morphology, as well as their corresponding SLN dispersions, free of
Lecithin- Short spacing Polymorphic
compounds, with differentiated aspects. SLNα presented greater
based SLNα Form
5.2 4.6 4.2 3.8 3.7 2.6 agglomeration and more evident crystallization confirmed by higher
FHPO day 1 5.5 4.7 4.1 3.9 2.6 β’ + β proportions of clusters than their controls, while maintaining electro­
(vw) (m) (s) (m) (w) static repulsion as shown by high ZP values. Higher clustering may be
FHPO day 7 5.5 4.7 4.3 3.9 2.6 β’ + β due to the α-tocopherol concentration used. Ying and Misran (2017)
(vw) (m) (s) (m) (w)
FHPO day 5.4 4.7 4.3 3.9 2.5 β’ + β
analyzed nanostructured lipid carriers (NLC) by microscopy and re­
15 (vw) (m) (s) (m) (w) ported that the higher α-tocopherol content, the greater the number of
FHPO day 5.4 4.6 4.2 3.8 2.5 β’ + β aggregates.
30 (vw) (m) (s) (m) (w)
FHPO day 5.3 4.6 4.2 3.8 2.5
3.6. Physical stability
β’ + β
60 (vw) (m) (s) (m) (w)
FHSO day 1 5.4 4.6 4,2 3.9 3.7 2.5 β’ + β
(vw) (s) (w) (m) (m) (w) Physical stability of the SLNα dispersions was analyzed in Turbiscan
FHSO day 7 5.3 4.6 4,2 3.9 3.7 2.6 β’ + β equipment, and TSI values indicated a loss of stability during the stor­
(w) (s) (w) (m) (m) (w) age. However, dispersions were not necessarily unstable with apparently
FHSO day 5.3 4.6 4,2 3.9 3.7 2.6 β’ + β
15 (w) (s) (w) (m) (m) (w)
visible destabilization, which requires TSI values above 10 (Silva et al.,
FHSO day 5.3 4.6 4,2 3.8 3.7 2.6 β’ + β 2011). TSI identifies changes in dispersions before their visibility to the
30 (w) (s) (w) (m) (m) (w) naked eye (Formulaction, 2021). Fig. 4 presents the TSI of SLNα dis­
FHSO day 5.3 4.6 4,2 3.9 3.7 2.6 β’ + β persions. Although the dispersions SLNα FHPO, SLNα FHSO, and SLNα
60 (w) (s) (w) (m) (m) (w)
FHCO lost stability over time, due to their unstable thermodynamic
FHCO day 1 5.4 4.7 4,2 3.8 2.5 β’ + β
(vw) (w) (m) (m) (w) characteristic, they presented TSI below 10 after 60 days of storage,
FHCO day 7 5.4 4.6 4,2 3.8 2.6 β’ + β which corresponds to direct analysis and visual observation. SLNα FHPO
(vw) (w) (s) (m) (w) showed a lower TSI value of 1.9 on day 60, followed by SLNα FHSO, and
FHCO day 5.3 4.6 4,2 3.8 2.5 β’ + β SLNα FHCO, with values of 3.4 and 4.2, respectively. Therefore, the
15 (vw) (w) (s) (m) (w)
FHCO day 5.3 4.6 4,2 3.7 2.5 β’ + β
increase in TAGs chain length of hardfats led to further destabilization. It
30 (vw) (w) (s) (m) (w) is worth mentioning that TSI values above 10 are reserved for largely
FHCO day 5.3 4.5 4,2 3.8 2.5 β’ + β unstable and visible systems (Formulaction, 2021).
60 (vw) (w) (s) (m) (w) Among the numerous emulsion destabilization mechanisms, floccu­
FHPO: fully hydrogenated palm oil; FHSO: fully hydrogenated soybean oil; lation was a possible change (results not shown) in the colloidal stability
FHCO: fully hydrogenated crambe oil. of SLNα, which is related to sedimentation, a physical mechanism that
*
Intensity: v. very; w. weak; m. medium; s. strong. was also observed in the corresponding SLN (Stahl et al., 2023).

6
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

Fig. 4. Turbiscan stability index (TSI) at 25 ◦ C of lecithin-based SLNα over the storage time (1 to 60d). FHPO: fully hydrogenated palm oil; FHSO: fully hydrogenated
soybean oil; FHCO: fully hydrogenated crambe oil, d: days.

However, all SLNα showed good stability with values lower than refer­ formed during the freeze-drying process. However, no significant loss of
ence values. Dispersion SLNα FHPO showed improved stability with α-tocopherol was observed during the study period. It is worth noting
lower values when compared to the control SLN (Stahl et al., 2023), that these results refer to the dispersions and can be related to the TSI,
while SLNα FHSO and SLNα FHCO showed higher TSI values when which showed low destabilization of the system over time, indicating
compared to the respective control SLN (Stahl et al., 2023). These results possible stability of bioactive compound.
indicate a possible preference of α-tocopherol for dispersions since a Although SLN may have been affected by α-tocopherol presence,
smaller hydrocarbon chain length of fatty acids usually indicates more emulsifier type and lipid used in the formulation may also have affected
polar molecules. TSI suggested that SLNα with smaller TAGs chain the particle behavior. The results of PS indicates that α-Tocopherol
length hardfats showed better dispersion stability and consequently, showed a preference for chain lengths similar to its structure and less
higher bioactive retention, with SLNα FHPO, SLNα FHSO, and SLNα polar lipids. Characterization of unloaded SLN was important for un­
FHCO described in increasing order of stability and TAGs chain length. derstanding transformations and identifying positive interactions. The
Another relevant aspect related to preference of α-tocopherol for SLNα is presence of α-tocopherol into SLN FHPO showed an effective ratio be­
their similar chemical composition. Tocopherols are formed by a basic tween the compounds, with lower destabilization over time. Results of
core consisting of a phenolic and a heterocyclic ring, linked to a satu­ α-tocopherol quantification showed the presence of compound in SLNα
rated 16-carbon side chain (Costa and Jorge, 2011), which resembles dispersions, indicating that both HPH technology and storage time at
palmitic acid that is also formed by a saturated 16-carbon chain. room temperature caused no loss of the compound.
SLNα has advantages over macro-scale compounds despite possible
loss of α-tocopherol due to the release caused by the organized crystal­
3.7. α-tocopherol stability lization of hardfats. Relkin et al. (2005) evaluated lipid nanoparticles
with high and low melting point TAGs when compared to conventional
Theα-tocopherol stability were determined on days 7, 15, and 30 of samples stored at room temperature for 8 weeks followed by heating at
storage, present in the formulation of SLN, to ensure that HPH did not 60 ◦ C for 3 h. Nanoparticles with high melting point TAGs showed lower
cause a loss of the compound. Table 2 presents the α-tocopherol stability vitamin loss when compared to the others.
of SLNα during 30 days of storage. Results showed a high deviation
between repetitions, mainly on day 7, probably due to the heterogeneity
4. Conclusion
of the samples since they were freeze-dried, and agglomerates may have
Fully hydrogenated palm, soybean and crambe oils proved to be lipid
Table 2 raw materials capable of producing α-tocopherol loaded SLN stable for
α-tocopherol stability of SLNα during 30 days of storage. physical and crystallization characteristics for up to 30 days.
(%) α- tocopherol Day 7 Day 15 Day 30 The presence of α-tocopherol altered the thermal behavior of the
SLNα FHPO 100a ± 5 91a ± 4 100a ± 2 particles, leading to increased crystallinity, with no changes in poly­
SLNα FHSO 83a ± 14 100a ± 2 100a ± 1 morphism and maintenance of SFC, when compared to the unloaded
SLNα FHCO 85a ± 17 100a ± 1 96a ± 4 solid lipid nanoparticles. These results corroborated with the higher
FHPO: fully hydrogenated palm oil; FHSO: fully hydrogenated soybean oil; enthalpy values and consequent crystallization disorder in the melting
FHCO: fully hydrogenated crambe oil. Different lowercase letters indicate a behavior and morphological analysis of the dispersions, which showed a
statistical difference between results in the same column. higher number of clusters. No specific behavior was observed for

7
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

particle size of the samples, which may have been affected by Dima, Ş., Dima, C., & Iordăchescu, G. (2015). Encapsulation of functional lipophilic food
and drug biocomponents. Food engineering reviews, 7(4), 417–438.
α-tocopherol, and was not relevant when considering absolute values.
Feng, J. L., Wang, Z. W., Zhang, J., Wang, Z. N., & Liu, F. (2009). Study on food-grade
Regarding ZP and SPAN values, no significant changes were observed in vitamin E microemulsions based on nonionic emulsifiers. Colloids and Surfaces A:
SLNα, when compared to respective SLN unloaded. Physicochemical and Engineering Aspects, 339(1–3), 1–6.
Overall, all α-tocopherol loaded solid lipid nanoparticles dispersions Formulaction. Smart scientific analysis. Available in: https://formulaction.com/wp-co
ntent/uploads/2022/08/TurbiscanLab_Brochure.pdf. Access in: 23/08/2021.
showed stability with no losses of α-tocopherol, promising results for Galli, F., Azzi, A., Birringer, M., Cook-Mills, J. M., Eggersdorfer, M., Frank, J., et al.
application in foods. The methodology used for obtaining SLNα proved (2017). Vitamin E: Emerging aspects and new directions. Free Radical Biology and
to be effective for incorporating α-tocopherol since there was no loss of Medicine, 102, 16–36.
Heurtault, B., Saulnier, P., Pech, B., Proust, J. E., & Benoit, J. P. (2003). Physico-chemical
compound in the dispersions. stability of colloidal lipid particles. Biomaterials, 24(23), 4283–4300.
Katouzian, I., & Jafari, S. M. (2016). Nano-encapsulation as a promising approach for
CRediT authorship contribution statement targeted delivery and controlled release of vitamins. Trends in Food Science &
Technology, 53, 34–48.
Klang, V., & Valenta, C. (2011). Lecithin-based nanoemulsions. Journal of Drug Delivery
Marcella Aparecida Stahl: Conceptualization, Methodology, Data Science and Technology, 21(1), 55–76.
curation, Writing – original draft. Fernanda Luisa Lüdtke: Methodol­ Kovacevic, A. B., et al. (2014). Solid lipid nanoparticles (SLN) stabilized with
polyhydroxysurfactants: Preparation, characterization and physical stability
ogy, Writing – review & editing. Renato Grimaldi: Methodology, investigation. Colloids and Surfaces A: Physicochemical and Engineering Aspects., 444,
Writing – review & editing. Mirna Lúcia Gigante: . Ana Paula Badan 15–25.
Ribeiro: Conceptualization, Funding acquisition, Supervision, Writing – Kumbhar, D. D., & Pokharkar, V. B. (2013). Engineering of a nanostructured lipid carrier
for the poorly water-soluble drug, bicalutamide: Physicochemical investigations.
review & editing, Validation. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 416, 32–42.
Lüdtke, F. L., Stahl, M. A., Grimaldi, R., Cardoso, L. P., Gigante, M. L., & Ribeiro, A. P. B.
(2022). High oleic sunflower oil and fully hydrogenated soybean oil nanostructured
Declaration of competing interest lipid carriers: Development and characterization. Colloids and Surfaces A:
Physicochemical and Engineering Aspects, 654, Article 130039.
The authors declare that they have no known competing financial Lüdtke, F. L., Grimaldi, R., Cardoso, L. P., Gigante, M. L., Vicente, A. A., &
Ribeiro, A. P. B. (2023). Development and Characterization of Fully Hydrogenated
interests or personal relationships that could have appeared to influence
Soybean Oil and High Oleic Sunflower Oil β-carotene Loaded Nanostructured Lipid
the work reported in this paper. Carriers. Food Biophysics.
Mohamed, A. S., & Mohamed, B. A. (2019). Nanotechnology in food systems: Application
and safety. World Journal of Advanced Research and Reviews, 2(2), 019–024.
Data availability
de Morais Ribeiro, L. N., Couto, V. M., Fraceto, L. F., & De Paula, E. (2018). Use of
nanoparticle concentration as a tool to understand the structural properties of
Data will be made available on request. colloids. Scientific reports, 8(1), 1–8.
Müller, R. H., Mäder, K., & Gohla, S. (2000). Solid lipid nanoparticles (SLN) for
controlled drug delivery–a review of the state of the art. European Journal of
Acknowledgments Pharmaceutics and Biopharmaceutics, 50(1), 161–177.
Nik, A. M., Langmaid, S., & Wright, A. J. (2012). Nonionic surfactant and interfacial
The authors are grateful to the Coordination for the Improvement of structure impact crystallinity and stability of β-carotene loaded lipid
nanodispersions. Journal of agricultural and food chemistry, 60(16), 4126–4135.
Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal Oehlke, K., Behsnilian, D., Mayer-Miebach, E., Weidler, P. G., & Greiner, R. (2017).
de Nível Superior - CAPES) for the financial support. Marcella Aparecida Edible solid lipid nanoparticles (SLN) as carrier system for antioxidants of different
Stahl and Fernanda Luisa Lüdtke thanks FAPESP (grants #18/03172-0, lipophilicity. PloS one, 12(2).
Oliveira, M. S., Mussi, S. V., Gomes, D. A., Yoshida, M. I., Frezard, F., Carregal, V. M.,
#19/05176-6, #16/11261-8 and 19/27354-3, São Paulo Research et al. (2016). α-Tocopherol succinate improves encapsulation and anticancer activity
Foundation - FAPESP). Ana Paula Badan Ribeiro thanks the National of doxorubicin loaded in solid lipid nanoparticles. Colloids and Surfaces B:
Council for Scientific and Technological Development (Conselho Biointerfaces, 140, 246–253.
Pacifico, S., Scognamiglio, M., D’Abrosca, B., Monaco, P., & Fiorentino, A. (2012).
Nacional de Desenvolvimento Científico e Tecnológico – CNPq) for the Tocopherols, tocotrienols, and their bioactive analogs. Handbook of analysis of active
productivity grant (#303429/2018-6). compounds in functional foods, 165.
Qian, C., Decker, E. A., Xiao, H., & McClements, D. J. (2013). Impact of lipid nanoparticle
physical state on particle aggregation and β-carotene degradation: Potential
Appendix A. Supplementary material limitations of solid lipid nanoparticles. Food Research International, 52(1), 342–349.
Relkin, P., & Sourdet, S. (2005). Factors affecting fat droplet aggregation in whipped
Supplementary data to this article can be found online at https://doi. frozen protein-stabilized emulsions. Food Hydrocolloids, 19(3), 503–511.
Ribeiro, A. P. B., Basso, R. C., & Kieckbusch, T. G. (2013). Effect of the addition of
org/10.1016/j.foodchem.2023.138149. hardfats on the physical properties of cocoa butter. European Journal of Lipid Science
and Technology, 115(3), 301–312.
References Saez, V., Souza, I. D. L., & Mansur, C. R. E. (2018). Lipid nanoparticles (SLN & NLC) for
delivery of vitamin E: A comprehensive review. International journal of cosmetic
science, 40(2), 103–116.
AOCS, D. F. (2009). Official methods and recommended practices of the American Oil
Salminen, H., Goemmel, C., Leuenberger, B. H., & Weiss, J. (2016). Influence of
Chemists’ Society. AOCS, 6ed, Champaign.
encapsulated functional lipids on crystal structure and chemical stability in solid
Averina, E. S., Müller, R. H., Popov, D. V., & Radnaeva, L. D. (2011). Physical and
lipid nanoparticles: Towards bioactive-based design of delivery systems. Food
chemical stability of nanostructured lipid drug carriers (NLC) based on natural lipids
chemistry, 190, 928–937.
from Baikal region (Siberia, Russia). Die Pharmazie-An International Journal of
Shah, R., Eldridge, D., Palombo, E., & Harding, I. (2014). Optimisation and Stability
Pharmaceutical Sciences, 66(5), 348–356.
Assessment of Solid Lipid Nanoparticles using Particle Size and Zeta Potential.
Bahari, L. A. S., & Hamishehkar, H. (2016). The impact of variables on particle size of
Journal of Physical Science, 25(1).
solid lipid nanoparticles and nanostructured lipid carriers; a comparative literature
Shukat, R., Bourgaux, C., & Relkin, P. (2012). Crystallisation behaviour of palm oil
review. Advanced pharmaceutical bulletin, 6(2), 143.
nanoemulsions carrying vitamin E. Journal of thermal analysis and calorimetry, 108
de Carvalho, S. M., Noronha, C. M., Floriani, C. L., Lino, R. C., Rocha, G., Bellettini, I. C.,
(1), 153–161.
et al. (2013). Optimization of α-tocopherol loaded solid lipid nanoparticles by
Silva, A. C., González-Mira, E., García, M. L., Egea, M. A., Fonseca, J., Silva, R., et al.
central composite design. Industrial Crops and Products, 49, 278–285.
(2011). Preparation, characterization and biocompatibility studies on risperidone-
Costa, T., & Jorge, N. (2011). Compostos bioativos benéficos presentes em castanhas e
loaded solid lipid nanoparticles (SLN): High pressure homogenization versus
nozes. Journal of Health Sciences, 13(3).
ultrasound. Colloids and Surfaces B: Biointerfaces, 86(1), 158–165.
Dan, N. (2016). Transport and release in nano-carriers for food applications. Journal of
Silva, M. G., de Godoi, K. R. R., Gigante, M. L., Cardoso, L. P., & Ribeiro, A. P. B. (2022).
Food Engineering, 175, 136–144.
Nanostructured lipid carriers for delivery of free phytosterols: Effect of lipid
Danaei, M., Dehghankhold, M., Ataei, S., Hasanzadeh Davarani, F., Javanmard, R.,
composition and chemical interesterification on physical stability. Colloids and
Dokhani, A., et al. (2018). Impact of particle size and polydispersity index on the
Surfaces A: Physicochemical and Engineering Aspects, 640, Article 128425. https://doi.
clinical applications of lipidic nanocarrier systems. Pharmaceutics, 10(2), 57.
org/10.1016/j.colsurfa.2022.128425
Das, S., Ng, W. K., & Tan, R. B. (2012). Are nanostructured lipid carriers (NLCs) better
Songsurang, K., Praphairaksit, N., Siraleartmukul, K., & Muangsin, N. (2011).
than solid lipid nanoparticles (SLN): Development, characterizations and
Electrospray fabrication of doxorubicin-chitosan-tripolyphosphate nanoparticles for
comparative evaluations of clotrimazole-loaded SLN and NLCs? European Journal of
delivery of doxorubicin. Archives of pharmacal research, 34, 583–592.
Pharmaceutical Sciences, 47(1), 139–151.

8
M. Aparecida Stahl et al. Food Chemistry 439 (2024) 138149

Stahl, M. A., Lüdtke, F. L., Grimaldi, R., Gigante, M. L., Ribeiro, A. P. B. (2023). Yang, Y., & McClements, D. J. (2013). Encapsulation of vitamin E in edible emulsions
Characterization and stability of solid lipid nanoparticles produced from different fabricated using a natural surfactant. Food Hydrocolloids, 30(2), 712–720.
fully hydrogenated oils. Food Research International (In Press). Yang, Y., Corona, A., III, Schubert, B., Reeder, R., & Henson, M. A. (2014). The effect of
Teixeira, M. C., Severino, P., Andreani, T., Boonme, P., Santini, A., Silva, A. M., et al. oil type on the aggregation stability of nanostructured lipid carriers. Journal of colloid
(2017). D-α-tocopherol nanoemulsions: Size properties, rheological behavior, and interface science, 418, 261–272.
surface tension, osmolarity and cytotoxicity. Saudi pharmaceutical journal, 25(2), Ying, L. Q., & Misran, M. (2017). Rheological and physicochemical characterization of
231–235. alpha tocopherol loaded lipid nanoparticles in thermoresponsive gel for topical
ur Rahman, U., Sahar, A., Ishaq, A., & Khalil, A. A. (2020). Design of Nanoparticles for application. Malaysian Journal of Fundamental and Applied Sciencs, 13, 248–252.
Future Beverage Industry. In Nanoengineering in the Beverage Industry (pp. 105-136). Ziani, K., Fang, Y., & McClements, D. J. (2012). Encapsulation of functional lipophilic
Academic Press. components in surfactant-based colloidal delivery systems: Vitamin E, vitamin D,
Wang, J. L., et al. (2014). Preparation and characterization of novel lipid carriers and lemon oil. Food chemistry, 134(2), 1106–1112.
containing microalgae oil for food applications. Journal of food science, 79(2),
E169–E177.