nanoencapsulation of food ingredients: from macromolecular ...€¦ · nanoencapsulation of food...

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


a simple story

about a simple concept

that simply works

Functional foods:

Smart protection and delivery of bioactives


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


The system of interest:

Amylose molecular complexes with LMW compounds


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


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:


Alkali solution

Acidification

DMSO

solution

Dilution into

water

Complexes


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,

…



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



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


What about the size?

With amylose: ~400 nm particles


AFM images (4.5μmX4.5μm) of V-amylose nanocapsules hosting

Linoleic Acid produced via DMSO method [A] or via acidification [B].

Outline


• What is the effect of the guest

molecule?


compounds?



• Conclusion


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


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.



A clear difference in FA’s mobility in the complex

Zabar, Lesmes, Schmidt, Shimoni, Bianco-Peled, Food Hydrocolloids (2009)


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:


0

0.5

2-6

6-72 Potential colonic delivery

Time [hr]


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!



Outline




compounds?



• Conclusion


Other nutrients / bioactives? – polyphenols?


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


Genistein release in simulated stomach

• PBS: pH=6.9, 37 ºC, 24h.

• HCl: pH=2, 37 ºC, 2h.


stomach

(Stomach simulated conditions) (control)


(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.



Kinetics of Genistein Release in

Simulated Intestinal Conditions

HACS


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


100nm 10mm

Cohen et al., J. Agric. Food Chem. 2008.

Colloidal stability

28

Outline




compounds?



• Conclusion



The test-tube

concept works!

But it is only a

test tube…

30


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


What’s now?

Development of foods containing nanoencapsulated ingredient

Enhanced bioavailability Enhanced bioavailability

http://www.unina.it/

http://www.karmat.co.il/

http://www.technion.ac.il/

http://www.hacettepe.edu.tr/

http://www.antonioamato.it/

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


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)


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


Total ALA 90°C

Omega-3 profile of ‘Flax seed oil’ complexes during high

temperature treatment 60⁰C (A) and 90⁰C (B).


Shelf life testing


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


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


Enzymatic digestion test


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




compounds?



• Conclusion


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


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


Outline




compounds?



• Conclusion


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.


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


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


[email protected]

www.technion.ac.il/~eshimoni

nanoencapsulation of food ingredients: from macromolecular ...€¦ · nanoencapsulation of food...

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