Solid Lipid Nanoparticles: A New
and Effective Delivery System for
Bioactives in Foods
Jochen Weiss*
Julian McClements, Thrandur Helgason, Tarek Awad, Eric Decker
*Food Structure and Functionality Laboratories
Department of Food Science and Biotechnology
University of Hohenheim
Garbenstrasse 25, 70599 Stuttgart, Germany
IFT International Food Nanoscience Conference
June 6th, 2009, Anaheim, CA
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Presentation Overview
• Foreword
• Solid Lipid Nanoparticles (SLN)
– What are they?
– How are they manufactured?
• Properties
P
ti and
dP
Problems
bl
off SLN
– Polymorphic Transitions
– Gelation
• Stabilization of SLN
– Pre-Crystallization
– Post-Cryzstallization
• Application Examples
– Omega-3 Encapsulation
– b-Carotene Encapsulation
• Conclusions
Foreword:
The ‘Structural’ Food Science Revolution
The Lipid Family
Simple
Droplets
The Biopolymer Family
Multiple
Emulsions
Solid Lipid
Particles
The Surfactant Family
Coated
Droplets
Pickering
Emulsions
Many
more …
Improved understanding of nanoscalar
assembly processes have led to this
explosion
p
!!!
Adapted from Julian McClements
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I. What Are “Solid Lipid Nanoparticles” (SLN)??
liquid
lipid (oil)
Emulsion
exchange
degradation
Surfactant
Layer
lipophilic
p
compound
No exchange
Less degradation
solid
lipid
Solid Lipid
Nanoparticle
• Liquid lipid in
emulsion is replaced
by high melting point
lipid
y
or waxes
• Glycerides
suitable
• Typical medium size
ranges
g from 50 - 500
nm
• At small sizes, crystal
structures become
dependent on
surfactant and size
• Polymorphism
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Why Solid Lipid Nanoparticles?
•
•
•
•
•
•
•
Better control over release kinetics of encapsulated
compound
– Engineering via size and lipid composition
– Melting
M lti can serve as trigger
ti
Enhanced bioavailability of entrapped bioactives
Chemical protection of labile incorporated
compounds
Much easier to manufacture than biopolymeric
nanoparticles
– No special solvents required
– Wider range of base materials (lipids)
– Conventional emulsion manufacturing methods
applicable
Raw materials essential the same as in emulsions
Very high long-term stability
Application
pp
versatility:
y
– Can be subjected to commercial sterilization
procedures
– Can be freeze-dried to produce powdered
formulation
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Conventional Carrier
dc/dt
20-50 m
cs
Microcarrier
dc/dt
2 5 m
2-5
cs
Nanocarrier
dc/dt
cs
200 nm
Dissolution velocity
Saturation solubility
5
Manufacturing
g of SLN
• Three different approaches:
– Hot homogenization
 homogenization at elevated temperatures
– Hot microemulsification
 Formation
F
ti off microemulsion
i
l i att elevated
l
t d temperatures
t
t
– Cold homogenization
 Homogenization at low temperatures using milling processes
• Each process has advantages and disadvantages
• Selection of suitable process predominantly governed by
type
yp of compound
p
to be encapsulated
p
• Scale-up procedures vary greatly between the different
processes
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Melting of Carrier Lipid and
Dispersing of Bioactive
Production of SLN by
Hot Homogenization
Dispersion of Bioactive‐Lipid in Hot Surfactant Solution
•
Coarse Pre‐emulsion Formation (Ultraturax)
•
Microfluidization at T > Tm
Hot Oil‐in‐Water Nanoemulsions
•
•
•
Hot homogenization
g
can be carried out by
y high
g
pressure homogenizers or high intensity
ultrasound
Metal contamination a possibilty wit highintensity ultrasound  coated probe
Production of nanoemulsions at elevated
temperatures  requires ability to thermostat
the homogenization chambers
T i l lilipid
Typical
id contents
t t b
between
t
5
5-10%,
10%
successful production of up to 40% reported
3-5 passes at 500-1500 bars
Solidification by Controlled Solidification
by Controlled
Cooling
Solid Lipid
Nanoparticles
Note: Small particle size and presence of emulsifiers
retards lipid crystallization – sample may remain as
shelf-stable supercooled melt for months/years
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II. Properties and Issues Surrounding SLN …
Localization of bioactives?
Issues with SLN!
 Kinetic instabilities
 Crystal structure:
polymorphic
transitions
 SLN dispersion
stability:
y creaming
g
 Microphase
separations during
crystallization
 Loading & formulation
 A lot of Expertise is
needed
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Fatty Acid Chain
Crystal Structures of
Ti l
Triglyceride
id SLN
SLNs
End view
hexagonal
•
•
•
•
•
SLN structure d
depends
d on
underlying crystal structure of
matrix
Diff
Different
t possible
ibl association
i ti
configurations of individual chains
Gives rise to longitudinal stacking
off TAG molecules
l
l in
i lamellae
l
ll
, ’ and  crystals  hexagonal,
cubic and orthogonal crystals with
diff
different
t latices
l ti
spacing
i
Temperature profiles during
production and storage essential
4.15Å
cubic
4.1-4.2Å
orthogonal
4.6 Å
3.8 Å
’

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2L


3L
9
The Issue of Polymorphic
T
Transformations
f
ti
 When
When polymorphic transitions polymorphic transitions
occur, the lipid crystals rearrange to assume a more ordered state
 Ostwald
Ostwald’ss step rule states:
step rule states:
 Thermodynamically less stable phase are initially formed and a stepwise phase changes to
a stepwise phase changes to more stable phases follows
 Thus, the α‐form form
transitions to β’ and finally to β
β
y β
 These crystals have different morphologies!
Himawan, C., V.M. Starov, and A.G.F. Stapley, Advances in
Colloid and Interface Science, 2006. 122(1-3): p. 3-33.
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Why are Polymorphic Transitions a Problem?
Oiling off !!
5oC
Melting
30 min.
75oC
Fluid SLN at 5°C
Gel at 5°C
Coalesced Droplets
After the initial formation of SLN, the suspensions increasingly lose fluidity
due to particle aggregation. This gelation process is highly time and
temperature sensitive
ii
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Polymorphic Transitions Depend on Storage
Temperature
Stored at 5°C
Stored at 1°C
Storage
Storage
Helgason, T., et al., Journal of Food Hydrocolloids, 2007.
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Polymorphic Transitions Correlate Directly with
Increases in Gel Strength
1e+5
80
TSLN
60
Tco
1oC
5oC
o
10 C
1e+4
1e+3
Tc
G* [P
Pa*s]
HSLN/
HC (%)
1°C
5°C
10°C
40

1e+2
1e+1
1e+0

1e-1
20
1e-2
0
1e-3
0
20
40
60
80
100
120
140
0
Time (min)
20
40
60
80
Time (min)
The ratio of melt enthalphy of stable SLN (DHSLN) to melt enthalpy of coalesced/separated
droplets increases with increasing holding temperature indicating a more rapid polymorphic
).
transformation in SLN (( to )
This corresponds to a simultaneous increase in G’
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Proposed Mechanism of SLN
D t bili ti
Destabilization
Awad, T., et al., Food Biophysics, 2007; Helgason, T.,
et al., Journal of Food Hydrocolloids, 2007.
SLN destabilization occurs via a complex combination of
polymorphic transitions, morphological changes and aggregation
that eventually lead to coalescence upon heating
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Morphological Changes Due to Polymorphic Transitions
Have Been Observed byy Others
TEM of SLN Preparation after 1 year storage
•
•
Dubes et al, European Journal of Pharmaceutics and
Biopharmaceutics, 2003, Vol. 55, 279-282
Dramatic morphological changes
during storage have been observed
even
e
e in initially
a y sstable
ab e S
SLN
preparations after long-term storage
The influence of crystal form on
shape of crystallized lipid droplets
has been observed by Bunjes and
coauthors
 polymorph (platelets)
Needle-shape crystals
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Idealized core-shell particle
(e.g. -3 loaded TAG SLN
with TAG shell)
r2
r1
e.g. at R=0.5, rSLN~60 nm 
maximally allowed size to
maintain an RDA of 300 mg in
a 1 wt% emulsion made of
fishoil!
Ratio of S
Shell Volu
ume to Tottal Volume
e
A Last Issue: Loading Capacity…
1.2
1.0
~ SLN
Regime
R 
Vcore
Vtotal
4 3 4
3
 r2    r2  r 
3
3
4 3
 r2
3
 r  r 
 1 2
0.8
3
r23
0.6
~ Mi
Minimal
i l Loading
L di
Boundary
0.4
~Transparency Boundary
0.2
0.0
100
200
300
400
500
Particle Size (nm)
With decreasing size, the amount of material that can be loaded in the particle y
g
decreases. In Foods, this can be a severely limiting issue since RDAs (recommended daily allowances) must be delivered
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III. Approaches to Stabilization of SLN
- Modulation via Surfactant Choice • Choice of surfactants in formation of stable SLN critical:
– Initial crystal structure (pre
(pre-solidification):
solidification):
• Surfactants with liquid lipid tails will form a fluid membrane around
the solidifying lipids upon crystallization. In this case crystallization
is not initiated/aided by the surfactants.
• Surfactants with solid lipid tails may interact with the solidifying
lipid matrix and act as nuclei. At small droplet diameters, such
emulsifiers may have substantial impact on the resulting crystal
structure
– Polymorphic transitions (post-solidification)
• Surfactant concentration and type may have an influence on the
kinetics of polymorphic transitions after crystallization.
– Dispersion stability (post-solidification)
• Insufficient surfactant may result in aggregation of the dispersion
due to hydrophobic interactions
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Influence of Surfactant on Crystallization of SLN
(Pre-Solidification Influence)
•
•
•
DSC heating curves of SLNs after controlled
cooling
•
Use of long-chain fatty
acid containing
phospholipids
p
p
p
lowers
supercooling tendency
Solidification of PL prior
to TAG solidification
alters crystallization
b h i
behavior
Modification of Tc thus
possible through
appropriate choice of
emulsifier
General retardation of
polymorphic transitions
in the presence of
saturated and egg
lecithin
Bunjes and Koch, 2005, J. Cont. Release, Vol. 107, 229-243
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Influence of Surfactant Type on SLN Formation
(Tween 20
20, 40
40, 60 & 80) – Pre-Crystallization
Pre Crystallization
First Cooling Cycle
Second Cooling Cycle
Tween 80
Tween 80
Tween 60
Tween 60
Tween 40
Tween 40
Tween 20
Tween 20
Surfactant type influences the crystal structures generated!
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Modulation of Polymorphic Transitions by PostAddition of Surfactant
5% SDS
2,5% SDS
1% SDS
0,5% SDS
0,1% SDS
0,05% SDS
SDS
S Concenttration
• SLN were initially
manufactured
f t d with
ith
10% tripalmitin and
2% Tween 20
• Immediately after
homogenization
SDS was added
• Addition of SDS at
high concentration
increasingly
stabilized the α
α- and
β´- form
0 01% SDS
0,01%
0% SDS
30°C
30
C
40°C
40
C
50°C
50
C
60°C
60
C
70°C
70
C
Helgason, T., et al., Journal of Food Hydrocolloids, 2007.
Food Structure and Functionality
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Can Addition of Surfactants Post-Solidification
Help Stabilize the Dispersion?
Liquid
Solid
Added
Tween 20
(%)
d43
Stdev
d43
Stdev
d32
Stdev
d32
Stdev
0
0.770
0.085
0.163
0.006
Gel
X
Gel
X
0.01
0.677
0.051
0.160
0.000
Gel
X
Gel
X
0.025
0.837
0.412
0.163
0.006
Gel
X
Gel
X
0.05
0.680
0.046
0.163
0.006
Gel
X
Gel
X
0.075
0.683
0.012
0.163
0.006
Gel
X
Gel
X
01
0.1
0 950
0.950
0 471
0.471
0 163
0.163
0 006
0.006
G l
Gel
X
G l
Gel
X
0.5
0.783
0.159
0.167
0.006
Gel
X
Gel
X
1
0.643
0.136
0.163
0.006
9.187
6.430
0.197
0.015
25
2.5
0 990
0.990
0 546
0.546
0 163
0.163
0 006
0.006
7 413
7.413
4 924
4.924
0 193
0.193
0 015
0.015
5
0.997
0.197
0.167
0.006
4.077
1.269
0.193
0.006
Addition of surfactant appears
pp
to help
p stabilize the dispersion
p
Helgason et al., Langmuir, 2008 (in Print)
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Evidence of Additional Surfactant Adsorption Upon SolidLiquid
q
Transitions
Liquid
Solid
So
d
60
Liquid
Solid
3
Twaq/Tw
wTotal (%)
Tween 20 D
Detected (%)
4
2
1
50
40
30
20
0
0
1
2
3
4
5
6
Tween 20 Added (%)
7
10
0
1
2
3
4
5
6
7
TwTotal Concentration (%)
Solidification of droplets results in decreases in Tween 20 in the aqueous phase,
gg
g additional absorption
p
of the surfactant to the newly
y formed interfaces
suggesting
Helgason et al., Langmuir, 2008 (In Print)
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•
•
•
In the presence of
excess surfactant
f t t
(2/6 wt%), particles
grew upon
solification but did
solification,
not aggregate
In this case,
dispersion
remained stable
If insufficient
surfactant was
present, particles
aggregated rapidly
upon cooling
Hyydrodyn
namic Radius (n
nm)
Crystallization in the Presence of Excess
Surfactant
1000
900
800
700
Aggregation
1% Tween 20 added
2% Tween 20 added
6% Tween 20 added
600
500
Cooling
400
300
200
C t lli ti
Crystallization
Stable Dispersion
100
5
10
15
20
25
30
35
40
Temperature (°C)
( C)
Helgason et al., Langmuir, 2008 (In Print)
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What About Crystal Structures?
(Post-Solidification)
Heating enthalpy of tripalmitin SLN
after addition of Tween 20 after
storage for 24 hours at 20°C
20
Cooling enthalpy of tripalmitin SLN
after addition of Tween 20 after melting
at 75°C
5%
5%
2 5%
2.5%
2.5%
1%
1%
0.1%
0 1%
0.1%
0.05%
0.05%
0.01%
0.01%
0%
0%
30
40
50
60
70
20
30
40
50
60
70
At increased added Tween 20 concentrations, more complex melting behavior suggesting alternative
crystal structures
Helgason et al., Langmuir, 2008 (In Print)
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Proposed Mechanisms of Surfactant Modulation
• Pre-solidification:
Pre solidification:
– Surfactants may act as seeds for the crystallization depending on their
molecular structure (liquid/solid tails) and the droplet size (no clear
boundary, gradual modifications of crystal structures apparent)
– Sufficient surfactants must be available to form the liquid dispersion –
which is less than the conc. required for solid dispersions
Liquid
q
Tail Surfactants
d < ~150 nm
Solid Tail Surfactants
d >> ~150 nm
d < ~150 nm
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d >> ~150 nm
25
Proposed Mechanisms of Surfactant Modulation
• Post-solidification:
– Surfactants can aid stabilization of SLN dispersions by (a)
modulating
g polymorphic
p y
p
transitions and (b)
( ) stabilizing
gg
generated
crystals
At low surfactant concentration
Liquid lipid
At increased surfactant concentration
Low/no excess surfactant
f
Cool to 5°C
Crystallization
Addition of Surfactant
Cool to 5°C
Crystallization
Excess surfactant
f
Solid lipid
Polymorphic
transitions, uncovered
surfaces,
f
aggregation
i
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Increased surface, excess surfactant adsorbs to interface
26
V. Application Examples
Case 1: Omega
Omega-3
3 Fatty Acids
80
1.00
0.10
0.05
0 00
0.00
o
0.25
Tc,Tm ( C)
Heat flow
w (J/g)
0.05
y = -15.936x
15 936x + 40.037
40 037
R2 = 0.9765
70
-3 fatty acid

d content
0.75
Melting
60
Melt temperature
50
40
Crystallization temp.
30
y = -10.855x 2 - 1.569x + 64.069
R2 = 0.9954
20
10
10
20
30
40
50
o
60
70
0
Temperature ( C)
02
0.2
04
0.4
06
0.6
08
0.8
1
 
In bulk tripalmitin in the presence of -3 fatty acids – significant
d
decreases
iin melting
lti and
d crystallization
t lli ti ttemp (50% lloading
di
d
desired)
i d)
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How Does This Affect Production of SLN???
Without Fish Oil
5
Cool1
5
Heat
Cool2
2.5
Heat flow ((J/g)
Heat flow (JJ/g)
H
Tween
10 20 Stabilized
With 0.25% Fish Oil
0
-5
-10
Cool1
Heat
Cool2
0
-2.5
25
-5
-7.5
-10
10
-12.5
-15
-15
0
20
40
o
60
80
0
Temperature ( C)
20
40
o
60
80
Temperature ( C)
Formation of -crystals suppressed, formation of thermodynamically
stable
t bl  promoted.
t d
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Dispersion Stability of SLN in the Presence of 3 Fatty Acids
200
190
Z-ava
arege size (nm)
• Cr
Crystallized
stalli ed
nanoemulsion with
>25% w-3 fatty acids
DO NOT aggregate
• Indicates that
morphological
changes associated
with polymorphic
transitions are
suppressed.
180
170
0% -3
10% -3
25% -3
160
150
140
130
0
10
20
30
40
50
60
Time (min)
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Rheology
gy of SLN Containing
g -3 Fatty
y Acids
1 E+04
1.E+04
G* (Pa))
• -3
 3 fatty acid
containing SLN did
not show a
noticeable increase
in complex modulus
• The sample
remained
i d fluid
fl id d
during
i
the first cooling
process and also
d i a subsequent
during
b
t
additional heating
and cooling cycle.
+0.00
+0 25
+0.25
+0.25 (melting)
1.E+02
1.E+00
1.E-02
0
20
40
60
80
100
o
Temperature ( C)
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Potential Structure of SLN Containing -3
Fatty Acids
0% ω-3
Solid lipid
Crystallization
Liquid lipid
Tripalmitin
crystal covered
y surface -3
by
fatty acids
>25% ω-3
Liquid oil
inside the
crystal matrix
retards the
shape change
Crystallization
y
Actual structure as yet unkown!!!
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Tripalmitin
crystal
t l
containing
microp
-3
dispersed
fatty acids
31
Case 2: -Carotene in SLN
Liq. Surf.
Miglyol
& -carotene
(0.1%)
3-6
3
6 g SLN/day
Liquid Matrix
Translates to:
Solid
d Matrix
RDA:
3-6 mg-day
-carotene
Solid Surf.
Liq. Surf.
Tripalmitin &
-carotene
(0 1%)
(0.1%)
HLPPP
Hydrogenated
lecithin
Main surfactant
(w/w)
2 4% Phospholipon
2.4%
80H
ULPPP
Unsaturated lecithin
2.4% Alcolec PC 75
Tw60PPP
Tween 60
1.4% Tween 60
Tw80PPP
Tween 80
1.4% Tween 80
HLM
Hydrogenated
lecithin
2.4% Phospholipon
80H
ULM
Unsat rated lecithin
Unsaturated
2 4% Alcolec PC 75
2.4%
Tw60M
Tween 60
1.4% Tween 60
Tw80M
Tween 80
1.4% Tween 80
Code
Surfactant system
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Solid Surf.
Co-surfactant
Lipid (w/w)
(w/w)
0 6%
0.6%
10%
Taurodeoxycholate Tripalmitin
0.6%
10%
Taurodeoxycholate Tripalmitin
0.6%
10%
Taurodeoxycholate
y
Tripalmitin
p
0.6%
10%
Taurodeoxycholate Tripalmitin
0.6%
10%
Taurodeoxycholate
Miglyol
0.6%
10%
Taurodeoxycholate
Miglyol
0.6%
10%
Taurodeoxycholate
Miglyol
0.6%
10%
Taurodeoxycholate
Miglyol
32
Crystallization of SLN with -carotene
TW60PPP
Heat Flow
F
TW80PPP
Exoth
hermal
Using surfactants
that solidify prior
to the matrix
increases the
crystallization
temperature of
the SLN
The system with
hydrogenated
lecithin
crystallized at the
highest
temperature
0
Onset 21.0°C
ULPPP
Onset 21.1°C
Onset 30.3°C
HLPPP
10
Onset 24.4°C
20
30
40
Cooling
50
60
Temperature (°C)
g
g p of the initial cooling
g of
Figure
1. DSC thermographs
different surfactant system
Technologie Funktioneller
33
Lebensmittel
70
Melting Analysis of SLN
TW80PPP
Heat F
Flow
TW60PPP
Exo
othermal
Melting peak at 40ºC
indicates presence of αsub-cell crystals
Present in high-melting
surfactant-stabilized
particles
More complex melting
indicated more complex
crystal structure
Surface initiated
crystallization?
Increased rigidity of the
interface?
Onset 40.9
40 9°C
C
ULPPP
HLPPP
Onset 42.1°C
20
30
40
Heating
50
60
70
Temperature (°C)
Figure 2.
Melting thermographs after 1 day of
storage
att 20°C for
t
f SLN with
ith carotene
t
Technologie Funktioneller
34
Lebensmittel
80
No aggregation or
gelation was observed
Hydrodynamic radius
increased but much
less so in SLN that
had been
manufactured with
hi h melting
high
lti
surfactants
Hydro
odynamic Radius (n
nm)
What About Gelation and Shape Changes?
200
33.4% incr.
180
H LPPP
U LPPP
Tw 80PPP
Tw 60PPP
21.9% incr.
160
Cooling
18.5% incr.
140
2.8% incr.
120
0
10
20
30
40
50
Tem perature (°C)
Apparently, SLN remain
spherical with solid surfactants
Figure 4. Size increase of all surfactant systems,
during cooling from 45-5°C.
45 5 C.
Technologie Funktioneller
35
Lebensmittel
Measured as
relative decrease in
concentration
Dramatic
improvement in
stability of βcarotene in HLPPP
systems
t
Tween 60 performed
better than Tween
80, but less well
than phospholipids
Rel.--Caroten
ne Conte
ent (%)
β-Carotene Stability in SLN
120
100
80
HLPPP
ULPPP
Tw80PPP
Tw60PPP
60
40
20
0
0
5
10
15
20
25
o
Storage Time at 20 C (Days)
Figure 5.
β-carotene breakdown over time at 20°C,
using tripalmitin as an lipid matrix
Technologie Funktioneller
36
Lebensmittel
Mechanism of Bioactive Stabilization in SLN
β -carotene
Crystallization
and storage
β-carotene is
expelled
ll d when
h th
the
particle transitions to
achieve a
y
y
thermodynamically
more favorable form
Liquid Surfactant
Crystallization
and storage
β -carotene
Particle
crystallizes in a
crystal form that
is well suited to
maintain the β carotene
dispersed
Solid Surfactant
Technologie Funktioneller
37
Lebensmittel
V. Conclusions
Co c us o s
• SLN are a promising nanoscaler delivery system for the
food
ood industry
dus y due to
o the
e fact
ac that:
a
–
–
–
–
Large scale production possible, no organic solvents needed
High concentrations of functional compounds can be achieved
Lyophilization possible
Spray drying for lipids with Tm > 70ºC to yield powders
• Solid lipid nanoparticles are non-trivial systems with
potentially complex structures that include variations in
– Particle morphology
morphology,
– Internal particle microstructure
– Internal crystal structure
• Manufactures need to consider:
– Lipid matrix compositional changes upon inclusion of bioactive
– Choice of surfactant!!!!
– Manufacturing conditions
Food Structure and Functionality
Laboratories
38