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ncapsulation of olyunsaturated Fatty Acid sters
with olid Lipid articles
onald
olser
ussell esearch Center, Agricultural esearch Service, United States Department of Agriculture, Athens, Georgia, USA.
Corresponding author email: [email protected]
Abstract: Encapsulation of structurally sensitive compounds within a solid lipid matrix provides a barrier to prooxidant compounds
and effectively limits the extent of oxidative degradation. This offers a simple approach to preserve the bioactivity of labile structures.
The technology was developed for cosmetic and pharmaceutical products but may be applied to additives used in food and feed
formulations. The encapsulation of docosahexaenoic acid (DHA) and α-linolenic acid (ALA) was examined as model compounds of
current interest in functional foods and feeds. Solid lipid particles were prepared from triglycerides containing saturated and unsaturated
fatty acids and evaluated by differential scanning calorimetry. The thermal characteristics of the lipids used to form the particle were
related to molecular structure and could be adjusted by selection of the appropriate component fatty acids. Encapsulation by solid lipid
particles provides a method to inhibit oxidation and improve shelf life of products formulated with DHA and ALA.
Keywords: calorimetry, docosahexaenoic acid, linolenic acid, nanoparticles, oxidative stability
Lipid Insights 2012:5 1–5
doi: 10.4137/LPI.S7901
This article is available from http://www.la-press.com.
© the author(s), publisher and licensee Libertas Academica Ltd.
This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited.
Lipid Insights 2012:5
1
olser
Introduction
The health benefits associated with the polyunsaturated acids such as α-linolenic acid (ALA) and docosahexaenoic acid (DHA) have generated interest in
formulating foods and dietary supplements with these
compounds. However, the highly unsaturated structure
of these compounds is prone to oxidation and loss of
bioactivity. Encapsulation techniques can inhibit degradation and preserve the quality of products formulated with these compounds.1,2 The capsule or coating
acts as a barrier to prevent reaction of the encapsulated
compound. The capsule may also be designed with
additional chemical functionality that provides binding sites for cellular recognition, response to changes
in pH conditions, or additional protective groups.3,4
Solid lipid particles consist of a solid lipid matrix
with the bioactive component dispersed throughout
the solid phase. There are a variety of techniques used
to manufacture these materials including high pressure homogenization and solvent diffusion.5–9 The
first method requires more mechanical energy input
than the second method but does not depend on the
use of an organic solvent which can be a limitation
for certain products or markets. Solid lipid particles
found initial applications for the delivery of therapeutic agents and represented an alternative to the
use of liposomes for the encapsulation of bioactives.
Pharmaceutical applications for solid lipid particles
include oral, parenteral, and topical drug delivery.7,10–18
Advantages include improved bioavailability, controlled release, and stabilization. Limitations include
drug loading capacity and reports of drug expulsion
during storage. However, these properties are related
to compatibility between the solid lipid matrix and
the bioactive compound which can be controlled by
careful selection of the lipid matrix.
The current study is motivated by the development
of solid lipid particles for food and feed applications.
The primary benefit expected from the use of these
materials is the increased stability of the encapsulated
compound leading to extended shelf life of formulated
products. The selection of triglycerides for such application is determined by melting point range which may
be adjusted by blending the appropriate structures to
provide the desired property for the specific application.
A secondary benefit can be realized in terms of sensory
properties where adverse flavour attributes of the encapsulated compound may be masked by the lipid matrix.
2
This report describes how lipid particles were prepared
from binary mixtures of saturated and unsaturated triglycerides and used to encapsulate omega 3 fatty acid
esters. Thermal properties of the lipid particles were
characterized by differential scanning calorimetry. The
physical stability of the particles was examined by particle size analysis and the chemical stability of encapsulated material was monitored by infrared spectroscopy
while heating to induce degradation.
Materials and Methods
Preparation
Lipid standards used in this investigation were at least
99% pure and included docohexaenoic acid methyl
ester, linolenic acid methyl ester, palmitic acid methyl
ester, stearic acid methyl ester, trimyristin, triolein, tripalmitin, and tristearin (Nu-Check Prep, Inc., Elysian,
MN USA). Tween 80 (Fisher Scientific, Fair Lawn, NJ,
USA) was used as the surfactant. Encapsulated materials were prepared by heating a mixture of the selected
lipids together and generating the corresponding oil-inwater emulsion. A typical experiment combined 50 mg
tripalmitin and 50 mg tristearin with 10 mg docohexaenoic acid methyl ester. The lipids were heated at
75 °C for 2 minutes. A 4 mL volume of aqueous Tween
80 (0.5 wt%) was quickly added to the melted lipids
and homogenized for 90 seconds at 20 K RPM with a
tissue homogenizer (Omni, Waterbury, CT, USA). This
emulsion was further processed by a laboratory microfluidizer (Microfluidics, Newton, MA, USA).
Characterization
Particle size distributions were measured with a
Nicomp model 380 ZLS particle size system (Nicomp
PSS, Santa Barbara, CA, USA). Measurements were
taken at 23 °C using a 635 nm source and a scattering angle of 90°. Samples were prepared for measurement by dilution in deionized water. Calorimetry data
were collected with a TA Instruments model Q20 differential scanning calorimeter (TA Instruments, New
Castle, DE, USA). The instrument was calibrated with
an Indium standard. Aluminium sample pans were
used and samples were scanned at 5 °C/min over the
temperature range of 25 °C to 90 °C.
Oxidative Stability Degradation of encapsulated
DHA was monitored by mid infrared (MIR) spectroscopy using attenuated total reflectance (ATR). Spectra
were collected with a Tensor 27 FT-IR system (Bruker
Lipid Insights 2012:5
ncapsulation of polyunsaturated fatty acid esters
Optics, Billerica, MA) equipped with a Model 300
Golden Gate diamond ATR and temperature controller (Specac, Ltd., London, England). A sample was
placed on the ATR crystal and heated from 25 °C
to 120 °C at 5 C°/min while spectra were collected
at 5 minute intervals. Samples were scanned over
the range 4000–600 cm−1 at a resolution of 4 cm−1. The
spectrum was the result of 128 co-added scans. The
instrument was controlled by OPUS v 5.0 software
and spectra were processed using Grams/AI v 8.0
(Thermo Fisher Corp., Waltham, MA, USA).
Results and Discussion
Lipid particles generated from fluidized mixtures
of saturated lipids such as tripalmitin and tristearin
or trimyristin and tripalmitin exhibited unimodal
particle size distributions with mean diameters of
277.6 ± 15.6 nm or 270.2 ± 13.8 nm, respectively.
Particles prepared from mixtures of saturated and unsaturated lipids such as tripalmitin and triolein exhibited
slightly smaller mean diameters of 220.1 ± 13.1 nm.
The particle size distributions were unchanged over
6 months storage at ambient conditions which demonstrates the physical stability of the lipid particles.
Thermal properties of these lipid particles were
measured by differential scanning calorimetry. Figure 1
compares results obtained for lipid particles prepared
from binary mixtures of the triglycerides. The melting points of the individual components measured
59.6 °C, 68.0 °C, and 74.5 °C for tristearin, tripalmitin,
and trimyristin, respectively. The melting points of the
solid lipid particles prepared from binary mixtures were
lowered approximately 5 °C by blending a saturated lipid
with the unsaturated lipid triolein. The melting point of
the lipid matrix used for encapsulation may be changed
in a predictable manner through selection of the appropriate mixture of lipid components. The performance of
the lipid particle used to form the matrix can be related
to structure and readily described in terms of melting
point. This offers a simple approach for product developers or formulators to exploit the melting properties of
solid fats and vegetable oil blends.
The influence of triglyceride structure on melting
point is clearly demonstrated in Figure 2 which shows
the variation in melting point for blends of tristearin and
soybean oil.19 Modification of tristearin by the introduction of an unsaturated fatty acid ester at the second
carbon of glycerol reduces the melting points for all
blend ratios. As the degree of unsaturation is increased
from one double bond in oleic to three in linolenic the
melting point is further reduced. Comparable results are
observed with tripalmitin (Fig. 3) but over a lower temperature range.20 Such structured triglycerides are typically prepared in small quantities for research purposes
and are not economical for low cost applications.21
However, commodity oils such as coconut and palm
that contain a high level of saturation and offer
similar opportunities to control the melting point of
mixtures. Figure 4 presents results obtained for blends
of these oils with soybean oil. These curves provide
guidance for product design and development. The use
of commercially available edible oils will facilitate the
adoption of this technology and avoid potential delays
associated with the regulatory process.
80
0
70
Temperature (°C)
Heat flow (W/g)
−5
−10
Trimyristin/Triolein
Tripalmitin/Triolein
Tristearin/Triolein
−15
60
SSS
SOS
SLS
SLnS
50
40
30
20
10
−20
20
30
40
50
60
70
80
90
Temperature (°C)
Figure 1. Melting behaviour of binary mixtures of triglycerides used in
particle matrix.
Lipid Insights 2012:5
0
20
40
60
80
100
Blend (wt%)
Figure 2. Mixtures of soybean oil with symmetrical triglyceride isomers
derived from tristearin (SSS) by substitution with oleic ( ; 9cis-18:1),
linoleic (L; 9cis, 12cis-18:2), or linolenic (Ln; 9cis, 12cis, 15cis-18:3).
3
olser
70
Encapsulated DHA
0.8
120 °C
0.6
50
PPP
POP
PLnP
40
Absorbance
Temperature (°C)
60
30
20
10
25 °C
0.4
DHA
120 °C
0.2
0.0
0
0
20
40
60
80
100
Blend (wt%)
4000
25 °C
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm )
−1
Figure 3. Mixtures of soybean oil with symmetrical triglyceride isomers
derived from tripalmitin (PPP) by substitution with oleic ( ; 9cis-18:1) or
linolenic (Ln; 9cis, 12cis, 15cis-18:3).
Figure 5. Spectra of docosahexaenoic acid methyl ester (D A) at 25 °C
and after heating to 120 °C compared to the corresponding spectra of
encapsulated D A. (Spectra separated for clarity.)
The oxidative stability of the encapsulated material
was investigated by heating samples of the lipid particles while collecting infrared spectra that would
reveal changes associated with degradation of the
unsaturated fatty acid esters. These investigations
were conducted on lipid particles composed of fully
saturated triglycerides such as myristin and palmitin
used to encapsulate DHA. This combination provided
the most sensitive and highly unsaturated lipid to be
tested with minimal interference from the capsule
lipids. Figure 5 displays the spectra of the DHA standard before and after heating. Prior to the heat treatment
the characteristic carbonyl stretching band is observed
at 1740 cm−1. Additional strong absorbances were evident at 703.6 cm−1, 1160 cm−1, 1436 cm−1, 2964 cm−1,
and 3013 cm−1. After heating to 120 °C the spectra
showed several spectral changes associated with oxidative degradation. The carbonyl stretch has shifted
to a lower wavenumber, from 1740 cm−1 to near
1730 cm−1, which is typical of an aldehyde rather than
an ester and a broad absorbance has appeared near
3500 cm−1 which is characteristic of an alcohol group.
Both of these changes are consistent with the expected
oxidation products and provide a useful measure of
degradation. This development occurred within
20 minutes of heating DHA that was not encapsulated
while encapsulated DHA did not show these changes.
The preparation of lipid particles is technically
straightforward and follows established procedures
used to manufacture cosmetic and pharmaceutical
products. Application of this technology to food and
feed products is expected to improve the stability of
additives and extend shelf life. The concern has been
expressed regarding the fate of nanoparticles in food
and feed formulations however the biodegradability
of lipids is well known and should not be an issue
for these triglycerides. Furthermore, formulating
mixtures of edible fats and oils will appeal to such
industries that have experience in processing lipid
ingredients. Additional topics to be explored include
the sensory properties of products containing lipid
particles. Although with the low additive levels projected for commercial products there would be minimal adverse effect expected and the potential benefit
of the particle to mask the flavour of the encapsulated
compound.
60
Temperature (°C)
50
40
30
20
Palm
Interesterified soy
Coconut
Cocoa butter
10
0
0
20
40
60
80
Blend (wt%)
Figure 4. Mixtures of soybean oil with other commodity oils.
4
100
Lipid Insights 2012:5
ncapsulation of polyunsaturated fatty acid esters
ummary
Solid lipid particles were prepared from binary triglyceride mixtures. The melting points of the mixtures
were lowered by blending a saturated lipid with triolein. The thermal behaviour of the lipid particle was
related to the triglyceride structure which offers a
simple approach for product design and development.
The biodegradability of these lipids and their ability
to stabilize bioactive compounds presents significant
advantages to the food and feed industry.
Acknowledgements
The author acknowledges Dr. Gary List for providing
data on structured triglycerides.
Disclosures
Author(s) have provided signed confirmations to the
publisher of their compliance with all applicable legal
and ethical obligations in respect to declaration of conflicts of interest, funding, authorship and contributorship, and compliance with ethical requirements in
respect to treatment of human and animal test subjects.
If this article contains identifiable human subject(s)
author(s) were required to supply signed patient consent
prior to publication. Author(s) have confirmed that the
published article is unique and not under consideration
nor published by any other publication and that they
have consent to reproduce any copyrighted material.
The peer reviewers declared no conflicts of interest.
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