nanoencapsulation of food ingredients: from macromolecular ...€¦ · nanoencapsulation of food...
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Nanoencapsulation of food ingredients:
From macromolecular nanostructuring
to smart delivery systems
Eyal Shimoni
Faculty of Biotechnology and Food engineering,
Russell Berrie Nanotechnology Institute,
Technion – Israel Institute of technology, Haifa, Israel.
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
a simple story
about a simple concept
that simply works
Functional foods:
Smart protection and delivery of bioactives
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
STABILITY Stable against heat, pH and
oxidation in food processing
TASTE & COLOR No unpleasant taste or color
SAFETY Mild on the stomach because of
its insolubility in gastric juices
BIOAVAILABILITY Sustained release, high
absorption and bioavailability
Outline
• Proof of concept – CLA
• What is the effect of the guest molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
The system of interest:
Amylose molecular complexes with LMW compounds
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Different # of Glucose per turn
6 7 8
In / Between the helixes
Oxidative &
Thermal stability
Stable in acid stomach
Released in
GI and colon Bioavailability
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
The concept:
Intimate guest-host interaction will prove useful, for…
P. Lebail et al., Carbohydrate
Polymers, 43 (2000), 317-326.
How do we make them:
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Alkali solution
Acidification
DMSO
solution
Dilution into
water
Complexes
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Lalush et al. Biomacromolecules, 2005.
Our model molecule: CLA
CLA: a group of polyunsaturated fatty acids that exist
as positional and stereoisomers of conjugated dienoic
octadecadienoate (18:2).
Physiological properties:
antiadipogenic,
antidiabetogenic,
anticarcinogenic,
antiatherosclerotic,
effects on bone formation,
…
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Lalush et al. Biomacromolecules, 2005.
XRD of amylose-CLA complexes
produced by two complexation methods
at 3 temperatures
water /DMSO Acidification
5 8 11 14 17 20 23 26 29 32
90C
60C
30C
5 8 11 14 17 20 23 26 29 32
30C
60C
90C
Inte
nsit
y
Inte
nsit
y
2oθ 2oθ
7.36
13.1
20.1
7.36
13.1
20.1
Thermal stability of amylose-CLA complexes:
DSC
Tcyst.
(oC)
Tonset (oC) Tm (
oC) ΔH (J/gr)
KOH / HCl 90 78.9 ± 2.8 89.6 ± 2.2 7 ± 4.1
KOH / HCl 60 90.8 ± 6.4 93.6 ± 4.1 12.5 ± 3.4
KOH / HCl 30 85.7 ± 0.4 89.9 ± 0.6 9.7 ± 1.2
DMSO 90 79 ± 5.1 94.1 ± 3.8 17.4 ± 2.7
DMSO 60 87.1 ± 5.2 92.3 ± 2.2 12.3 ± 4.1
DMSO 30 81.2 ± 0.9 88.9 ± 0.1 13 ± 2.1
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Lalush et al. Biomacromolecules, 2005.
60o
C
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30 40 50 60 70 80
Time (hr)
mo
le O
2 /
mo
le C
LA
0
1
2
3
4
5
6
7
8
9
10
90 60 30
water/DMSO KOH/HCl
Temperature (oC)
% R
ele
ased C
LA
0
20
40
60
80
100
120
140
control α-amylase β-amylase glucoamylase pancreatin
DMSO 90oC DMSO 60oC DMSO 30oC
Enzyme
% H
ydro
lysis
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
What about the size?
With amylose: ~400 nm particles
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
AFM images (4.5μmX4.5μm) of V-amylose nanocapsules hosting
Linoleic Acid produced via DMSO method [A] or via acidification [B].
Outline
• Proof of concept – CLA
• What is the effect of the guest
molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Change in degree of crystallinity
5 10 15 20 25 30
Bragg angle [2θ]
Inte
nsity [
Arb
. units]
5 10 15 20 25 30
Bragg angle [2θ]
(a) (b)
SA
SA
OA
LA
SA
LA
SA
OA
Polymorph 1
Polymorph 2
Polymorph 2
Polymorph 1
X-ray diffraction of V type amylose inclusion complexes hosting Stearic acid
(SA), Oleic acid (OA) and Linoleic acid (LA) made by acidification method (a) or
DMSO method (b). Crystalline polymorphism of V-amylose hosting stearic acid
denoted as polymorph 1 and polymorph 2.
Lesmes, Cohen, Shener, Shimoni, Food Hydrocolloids 23 (2009) 667–675
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
acidification method DMSO method
Different particle size
Particle size distribution curves of suspensions containing V-amylose inclusion
complexes hosting Stearic acid (SA), Oleic acid (OA) or Linoleic Acid.
Complexes were produced via DMSO method. Curves calculated based on the
light scattering of the samples processed using the general Fraunhofer model
weighted by volume.
Lesmes, Cohen, Shener, Shimoni, Food Hydrocolloids 23 (2009) 667–675
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
A clear difference in FA’s mobility in the complex
Zabar, Lesmes, Schmidt, Shimoni, Bianco-Peled, Food Hydrocolloids (2009)
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
180 160 140 120 100 80 60 40 20 ppm
Amylose-SA complexes
Amylose-LA complexes
Amylose-CLA complexes
13C CP/MAS solid state NMR spectra of amylose complexes hosting Stearic acid
(SA), Linoleic Acid (LA) and Conjugated Linoleic Acid (CLA) made DMSO method
at 90oC.
The time scale
is of interest:
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
0
0.5
2-6
6-72 Potential colonic delivery
Time [hr]
Lesmes, Cohen, Shener, Shimoni, Food Hydrocolloids 23 (2009) 667–675
Enzymatically induced release profile of stearic acid from two types of crystalline
polymorphs (termed V1 and V2) of V-type amylose nanocapsules prepared via DMSO
method. Controls were achieved by suspending samples in PBS for similar periods of time.
Values are expressed as replicate means (*signifies p<0.05; ** p<0.01; ***p<0.001).
Different release rate!
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Lesmes, Cohen, Shener, Shimoni, Food Hydrocolloids 23 (2009) 667–675
Outline
• Proof of concept – CLA
• What is the effect of the guest molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Other nutrients / bioactives? – polyphenols?
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Genistin Genistein
21
• Isoflavones - subclass in the flavonoids family.
• Beneficial health effects:
– Reducing the risk of cardiovascular diseases.
– Lowering rates of prostate, breast and colon cancers.
– Improving bone composition.
• Recommended dose: 40-50 mg daily*.
•J. Sesma . Essentials of functional foods, 2000.
X-ray Diffraction- HACS
HACS genistein
complexes
Amylose genistein
complexes
HACS no guest
V6III -?
Cohen. R., et. al., Journal of Agricultural and Food Chemistry, 56, 2008, 4212.
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 22
0
2
4
6
8
10
12
14
1/60 1/30 1/20 1/15 1/12 1/10
Ge
nis
tein
co
nte
nt
in c
om
ple
x*
GAR
115
82
54
30
14 13
0
20
40
60
80
100
120
1/60 1/30 1/20 1/15 1/12 1/10
Glu
co
se
ge
nis
tein
mo
lar
rati
o**
GAR
** Glucose genistein molar ratio = (amylose content in the complex/Mw (glucose))
(genistein content in the complex /Mw (genistein))
• Reported glucose-to-guest molar ratio: 8 - 25.
Glucose genistein molar ratio
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 23
Genistein release in simulated stomach
• PBS: pH=6.9, 37 ºC, 24h.
• HCl: pH=2, 37 ºC, 2h.
Cohen. R., et. al., Journal of Agricultural and Food Chemistry, 56, 2008, 4212.
stomach
(Stomach simulated conditions) (control)
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 24
(Stomach simulated
conditions)
(control) (Intestine simulated
conditions)
0
10
20
30
40
50
60
PBS HCL Pancreatin
Ge
nis
tein
rele
as
ed
(%
) HACS amylose
HCl
intestine
Genistein release in simulated intestinal
conditions
*Pancreatic solution: pH=6.9, 140 units/ml, 37 ºC, 24h.
Cohen. R., et. al., Journal of Agricultural and Food Chemistry, 56, 2008, 4212.
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 25
Kinetics of Genistein Release in
Simulated Intestinal Conditions
HACS
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 26
Atomic Force Microscopy (AFM) of
Genistein-Amylose Complexes Undiluted sample Diluted sample (X50)
500nm 500nm
4.5mm X 4.5mm; Tip: CSC21 No/Al – contact mode
• The aggregation is reversible.
• Mean particle size of 200 ± 90 nm. 27
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
100nm 10mm
Cohen et al., J. Agric. Food Chem. 2008.
Colloidal stability
28
Outline
• Proof of concept – CLA
• What is the effect of the guest molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
The test-tube
concept works!
But it is only a
test tube…
30
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Pilot scale manufacturing:
cheap starch + sub micron capsules
0
20
40
60
80
100
120
Blank α - amylase β - amylase glucoamylase pancreatin
Oxidative
& Thermal
stability
Colloidal
stability
Controlled
release
Nanosize
complexes
Acid
Alkali
Starch
Bioactive
Dual feed jet
homogenizer
Shimoni, Lesmes, Ungar. US Patent Application, 2006.
31
Proof of concept and some more…
• Gelatinization is critical.
• Significant size reduction.
• Controlled release.
Lesmes, Barchechath & Shimoni, Innovative Food Science and Emerging Technologies, 2008,
0
0.01
0.02
0.03
0.04
0.05
0.06
0 20 40 60 80 100
Particle diameter (µm)
Corn 25C dissol.
Corn 85C dissolBefore
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0 5 10 15 20 25
Particle diameter (µm)
After
4.4
57.0
0
20
40
60
80
100
PBS Pancreatin
% R
ele
as
ed
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences 32
What’s now?
Development of foods containing nanoencapsulated ingredient
Enhanced bioavailability Enhanced bioavailability
From Lab-scale to Pilot-scale processing
Preparation of Solution
Acidification
Drying of complexes
34 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
A1
B1
C1
A2
B2
C2
D1
E1
F1
D2
E2
F2
magnification 1000 (1) and 10000 (2) magnification, bar 100μm and 10μm respectively.
SEM – microparticles containing w-3
HACS
Flax oil
Spray
dried
Ethyl
Esters
Fish Oil
Fish Oil
Flax Oil
freeze
Dried
35 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Stability under different temperatures
Sample
Event 1 Event 2
Tonset (°C) Tmelting(°C) ΔH (J/g) Tonset (°C) Tmelting(°C) ΔH (J/g)
HACS
(processed) 85.5±1.3 100±1.6 2.41±0.7 125.7±10.8 131.7±10.3 1.67±0.9
Flax seed oil
pH=5.8 86.1±2.5 94.7±0.3 2.71±0.4 141.7±0.7 144.4±0.01 3.52±0.4
Ethyl esters
pH=5.9 82.6±1.3 94±0.6 2.08±1 139.1±1.5 144.3±1.2 1.64±0.5
Fish oil
pH=3.9 80.4±2 90.7±4.5 1.9±0.5 140.2±1.6 143.5±1.7 3.77±2.7
The onset temperature, melting temperatures and enthalpies of
DSC endotherms for different samples of HACS with and without
omega-3 All values were normalized to sample dry weight and expressed as mean±SD (n≥2)
36 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Stability under different temperatures
0
2
4
6
8
10
12
0 100 200
mg
ω-3
/10
0 m
g s
am
ple
time (min)
ALA 'Free core content' 60°C
ALA 'Core content' 60°C
Total ALA 60°C
0
2
4
6
8
10
12
0 100 200m
g ω
-3/1
00
mg
sa
mp
le
time (min)
ALA 'Free core content' 90°C
ALA 'Core content' 90°C
Total ALA 90°C
Omega-3 profile of ‘Flax seed oil’ complexes during high
temperature treatment 60⁰C (A) and 90⁰C (B).
37 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Shelf life testing
38 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Omega-3 profile of ‘Flax seed oil’ complexes during storage at
temperature 4⁰C, 25⁰C and 37⁰C
0
2
4
6
8
10
12
0 10 20 30
mg
ω-3
/10
0 m
g s
am
ple
time (days)
ALA 'Free core content'37°C
ALA 'Core content' 37°C
0
2
4
6
8
10
12
0 10 20 30
mg
ω-3
/10
0 m
g s
am
ple
time (days)
ALA 'Free core content'4°CALA 'Core content' 4°C
Total ALA 4°C
0
2
4
6
8
10
12
0 10 20 30
mg
ω-3
/10
0 m
g s
am
ple
time (days)
ALA 'Free core content'25°C
ALA 'Core content' 25°C
Enzymatic digestion test
39 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
0
20
40
60
80
100
pH=2, 2 hours pH=6.9, 3 hours pH=6.9 withAlpha-amylase
(50U/ml)
ω-3
rele
ased
(%
ou
t o
f to
tal)
Total ω-3
Amount of omega-3 present after simulated stomach conditions (pH=2, 2
hours), PBS (pH=6.9, 3 hours) and enzymatic digestion (α-amylose
(50U/ml), pH=6.9, 3 hours) in samples Flax seed oil pH=5.7
Outline
• Proof of concept – CLA
• What is the effect of the guest molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Animals, Diets and Treatment
10 male Sprague-Dawley rats (220-250g)
Rats were placed in metabolic cages and were fasted
overnight (free access to water).
after 12 h
HACS-genistein
30 mg genistein /kg of body
weight
HACS-genistein
30 mg genistein /kg of body
weight
41 Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
42
Food
intake
Feaces
Plasma
Tissue
Organ Kidney
Urine
Bioavailable
Higher in
complexed
genistein
Higher in
complexed
genistein
Lower in
complexed
genistein
HACS-genistein complexes increases genistein bioavailability
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Outline
• Proof of concept – CLA
• What is the effect of the guest molecule?
• Can we use the system for non FA’s
compounds?
• The continuous process
• Enhanced bioavailability
• Conclusion
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Conclusion
V-amylose offers the possibility of nanoencapsulating:
poly-unsaturated fatty acids, phenolic compounds, aroma
chemicals, and drugs.
Complexes form particles with diameter of 400nm-30mm.
Complexation enables pursuing an array of applications.
Using continuous up-scale process demonstrate the
feasibility of forming such systems from a cheap and
commercially viable food grade product.
Complexation enhanced the bioavailability of genistein in
rats.
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Technion
Biotech. & Food Eng.
Shimoni group A-Z:
Hagar Ades
Revital Cohen
Zoya Kaplun
Dr. Ellina Kesselman
Dr. Uri Lesmes
Dr. Ory Ramon
Dave Semyonov
Asher Shazman
Dr. Yael Ungar
Hebrew U., Rehovot Prof. Betty Schwartz
Supported by:
FP7
Niedersachsen-Israel
RBNI
ISF
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
Chemical Engineering
Prof. H. Bianco-Peled Keren Shamai
Shiran Zabar
University of Napoli Prof. V. Fogliano
Hacettepe Univ. Prof. V. Gokmen
Braunschweig Univ. Prof. Petra Mischnik Christian Boerk
45
Thank you
Prof. Eyal Shimoni
Faculty of Biotechnology and Food Engineering, &
Russel Berrie Nanotechnology Institute,
Technion – Israel Institute of Technology,
Haifa, Israel
Laboratory of Functional Foods, Nutraceuticals, and Food Nanosciences
www.technion.ac.il/~eshimoni