Nano/Micro Encapsulation Technologies Hydrocolloids Natural Semisynthetic Sulfated: Carrageenan, furcellaran, agar, iridophycan, fucoidan, hypnean Sulfated starch Carboxylic Alginate,

Lecture 3 (10/23/2011)

Nano/Micro Encapsulation Technologies

Qingrong Huang

Department of Food Science

Tel: 732-932-7193

Email: [email protected]

Food Delivery Systems

Motivations:

- Encapsulated materials can be protected from moisture,

heat, oxidation, or other extreme conditions;

- Enhance food stability and maintain viability;

- Some bad odors or tastes can be masked, etc…

Food Ingredients of Interest:

- Food flavors, enzymes, nutraceuticals, and food colors.

others: nutrients, food microbial, etc.

Encapsulation Basics

Encapsulation: a process by which one material or mixture of

materials is coated or entrapped within another material or system.

The material that is coated or entrapped can be a liquid, a solid

particle, or gas, and is referred as core material (or fill, internal

phase).

The material that forms the coating is referred to as wall material

(or carrier, shell, membrane, coating).

Common Encapsulation Methods

1. Spray Drying

• First, the carrier or wall material (such as maltodextrin, modified starch, gum,

etc…) is hydrated. The flavor or ingredient to be encapsulated is added to the

carrier and homogenized or thoroughly mixed into the system using a similar

technique. Typical ratio of carrier:core materials is 4:1.

• The mixture is homogenized to create small droplets of flavor or ingredient

within the carrier solution, and then fed into a spray dryer where it is atomized

through a nozzle or spinning wheel. Hot air contacts the

atomized particles and evaporates the water, producing a

dried particle that is a starch or carrier matrix containing

small droplets of flavor or core. The dried particles fall

to the bottom of the dryer and are collected.

Common Encapsulation Methods

1. Spray Drying

0

50

100

150

200

250

300

350

400

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 2,4

-Decad

ien

al Level (p

pm

)

Days of Shelf Life

Spray Dried 2,4-Decadienal Encapsulated in

Neobee; Shelf Life Study

5 Degrees Celcius 25 Degrees Celcius

40 Degrees Celcius 60 Degrees Celcius

Reference @ 40 Degrees Celcius Reference @ 60 Degrees Celcius

Common Hydrocolloids

Natural Semisynthetic

Sulfated:

Carrageenan, furcellaran, agar,

iridophycan, fucoidan, hypnean

Sulfated starch

Carboxylic

Alginate, pectin, Arabic, tragacanth,

karaya, ghatti, xanthan gum, quince seed,

larch, psyllium seed gum, okra gum, gellan

gum, flax seed gum

Carboxymethylcellulose, low

methoxylpectin, propylene glycol alginate,

triethanolamine alginate, carboxymethyl

locust bean gum, carboxymethyl guar gum

Phosphorylated

Phosphomannans Starch phosphate

Nonionic

Dextran, starch and its fractions, locust

beam gum, guar, tamarind seed gum,

laminaran

Methylcellulose, hydroxypropylcellulose,

hydroxyethylcellulose,

hydroxypropylmethylcellulose,

ethylhydroxyethylcellulose

SEM Images

(a) (b)

Characterization of Capsules

Optical image AFM height image

50 μm

Common Encapsulation Methods

1. Spray Drying

Advantages:

• Most economical and widely used method of encapsulation;

• Equipment is readily available and production cost is low;

• Also a dehydration process and used in the preparation of dried

materials such as powered milk;

Disadvantages:

• Producing very fine powder which needs further processing

such as agglomeration to instantized the dried material;

• Not good for heat sensitive materials.

Common Encapsulation Methods

2. Spray Cooling

• Similar to spray drying in that core material is dispersed in a liquified

coating or wall material and atomized, but unlike spray drying, there is

no water to be evaporated;

• The core and wall mixtures are atomized into cooled air which causes

the wall (typically a vegetable oil, melting point 45-120 °C) to solidify

around the core;

• this method is often used to encapsulate solid materials such as

vitamins or minerals;

• With the ability to select the melting point of the wall, this method of

encapsulation can be used for controlled release.

Common Encapsulation Methods

3. Media Milling (part 1)

• Media milling involves placing solid particles to be mechanically reduced to

nano dimensions in a ball mill or milling unit that contains milling media

beads. Dry and wet media milling procedures can be used.

• In a dry milling process, no solvent or liquid is added to the ball mill. In a

wet milling process a liquid is present. It usually is water, but can be a food

grade vegetable oil.

• In both dry and wet milling procedures, a dispersing agent is present. It may

be a polymer or nonpolymeric surfactant.

• Once loaded, the milling unit is rotated or agitated in some manner. Impact

and shearing forces between moving milling media beads reduce the

suspended solid particles to nanoparticles. Stress intensity coupled with

number of contact points with milling beads are primary factors that define

final milled particle size

Common Encapsulation Methods

3. Media Milling (part 2)

• Stress intensity is influenced by kinetic energy transmitted to the grinding

media through the agitator shaft within the machine’s stator housing. The

smaller the beads are, the smaller the nanoparticle produced, because the

number of contacts increases as bead size decreases. The rule of thumb is that

nanoparticle size equals 1/1000 the size of the grinding media;

• Milling media beads of 50-200 µm yield a fine particle size distribution.

Before wet-milled After wet-milled

Wet Milling System

Common Encapsulation Methods: 4.

Emulsion encapsulation/entrapment

• Key step: Formation of a oil-in-water (o/w) emulsion: The active

material to be encapsulated or entrapped is added to a hydrocolloid

solution. A small volume of this aqueous phase is then added to a large

volume of oil and the mixture is homogenized to form the emulsion.

Once the O/W emulsion is formed, the water-soluble polymer must be

insolubilized (cross-linked) to form tiny gels within the oil phase.

• The smaller the internal phase particle size of the emulsion, the

smaller the final microparticles will be. Cross-linking method: e.g.

alginate/Ca++.

2011-10-18 15

b-carotene:

Encapsulation of β-carotene using Polymer Micelles

antioxidant character, anticancer activity, enhancement of the

immune response, inhibition of mutagenesis, and blocking of free

radical-mediated reactions

The demand for b-carotene has increased.

2011-10-18 16

Background b-carotene:

lipophilic unsaturated

susceptible during storage

In order to insert the lipophilic b-carotene into aqueous

food systems and enhance its stability, different

technologies are investigated.

2011-10-18 17

Background Nanotechnology

serve as carriers for nutriceutical, effective vehicles for

drug delivery and controlled release, and gene therapy.

Encapsulation of b-carotene by nanotechnology

• Nanodispersion: protein, polymeric matrices

- B. S. Chu et al. J. Sci Food Agric 2008 88,1764-1769.

- S. C. Sutter et al. Interna. J. Pharmaceutics 2007 332, 45-54.

- C. P. Tan et al. J. Sci Food Agric 2005 85,121-126. (0.024%)

• Nanoparticle: emulsion, amphiphilic polymer

- M. Murakami et al. J. Chem Engin Japan 2008 41, 485-491.

- A. Hentschel et al. J. Food Sci 2008 73, N1-N6.

- X. Y. Pan et al. J. Colloid Interface Sci 2007 315, 456-463.

- J. P. Jee et al. Europ J Pharmac Biopharmac 2006 63 134-139

- S. A. Desobry et al. J. Food Sci 1997 62, 1158-1162.

2011-10-18 18

Background

Chitosan:

is a cationic polysaccharide

is a fully or partially N-

deacetylated product of naturally

abundant chitin

biocompatible, biodegradable, low-toxic, bioadhesive

attract increasing attention in the fields of food,

textile, cosmetics, biomedical, pharmaceutical,

and other industries.

2011-10-18 19

Background

poor solubility in

either water or

organic solvents

limited applications

Modified

Chitosan

hydrophilic

hydrophobic

A number of publications show the suitability of self-

assembled nanoparticles from modified chitosan for

encapsulation of sensitive ingredients.

Simultaneous Modification scarce

2011-10-18 20

Hypothesis and Objective

• Chitosan can be modified using acyl chloride as hydrophobic

group and MPEG* as hydrophilic group, which could exhibit

amphiphilic properties.

A hypothetical scheme of micellization of modified chitosan

• Because b-carotene is very lipophilic, the modified chitosan

nanoparticles can be used to encapsulate it, and to enhance its

solubility and stability.

* polyethylene glycol monomethyl ether

2011-10-18 21

Synthetic scheme of modified chitosan amphiphile:

The Structure

Nuclear Magnetic Resonance Fourier Transform Infrared

acylChitosan

acylChitoMPEG

2011-10-18 22

Sample

DS (Degree of Substitution)

NH2 NHAc NHR OR

chitosan 0.74 0.26 0 0

acylChitosan 0.69 0.23 0.08 0.92

Sample DSR=DS(NHR+OR) DSR/DSM DSM

acylChitoMPEG 1.0 2.18 0.46

Characterization:

Molecular formula of acylChitoMPEG C6H7O2(OH)1.08(OR)0.92(NH2)0.23(NHCOCH3)0.23(NHR)0.08(NHCOCH2CH2COMPEG)0.46

2011-10-18 23

Sample Milli-Q water Ethanol Acetone CH2Cl2 CHCl3 THF Dioxane

Chitosan - - - - - -

acylchitosan -

acylchitoMPEG

Solubility Test:

The improved solubility will make acylChitoMPEG be

easily fabricated into various micro- and/or nanoparticles,

which will extend the use of chitosan derivatives in

biomedical applications.

2011-10-18 24

Self-assembly Properties:

Critical Aggregation Concentration (CAC): 0.066 mg/mL.

CAC of chitosan: above 1 mg/mL. M. M. Amiji Carbohydr. Polym. 1995 26, 211-213

360 400 440 480 520

0

5

10

15

20

(a)

I3

I1

Inte

nsi

ty (

a.u

.)

Wavelength (nm)1E-3 0.01 0.1 1

0.90

1.05

1.20

1.35

1.50

1.65

1.80

1.95

(b)

acylChitoMPEG

Concentration (mg/mL)

I 1/I

3

2011-10-18 25

Surface morphology images of acylChitoMPEG.

Left is height image and right is phase image

Atomic Force Microscopy(AFM) Images:

2011-10-18 26

Single stretched exponential fit by dynamic light scattering (DLS)

Particle Size:

100 101 102 103 104 105 106

0.9

1.0

1.1

1.2

1.3

1.4

1.5

G (

q,t

)

t (s)

sample Diameter (nm)

by AFM by SEF

acylChitoMPEG 17.6 24.4

2011-10-18 27

Structure by Synchrotron small-angle X-ray scattering (SAXS):

10-4

10-3

10-2

10-1

100

I(Q

) (c

m-1

)

5 6 7 8

10-2

2 3 4 5 6 7 8

10-1

2 3 4 5 6 7 8

100

Q (Å-1

)

2.5x10-4

2.0

1.5

1.0

0.5

0.0

P(r

) (a

.u.)

200150100500

r (Å)

acylChitoMPEG 20 mg/mL

10 mg/mL

5 mg/mL

2.5 mg/mL

1.25 mg/mL

-2

MΔbnc=)=I(Q /0 2

I - scattering intensity

Q -scattering vector

c -sample concentration

n -association number

-scattering length

difference of one molecule

relative to the surrounding

medium

M -the molecular weight

b

The scattering profile

Slope: large scale

agglomeration of

associated particles

2011-10-18 28

0.1 1 10 100 1000

40

60

80

100

120

140

160

Rel

ativ

e C

ell

Via

bil

ity (

%)

Concentration (g/mL)

Cytotoxicity Analysis:

Suggesting that it was well biocompatible and had the potential to be used

in biomedical applications and encapsulation of active food ingredients.

2011-10-18 29

Picture of sample solutions encapsulating b-carotene:

Concentration of sample in water: 10 mg/mL

ε-Poly(lysine)-Based Micelles

ε-Poly(lysine), or EPL, is generated naturally

• EPL is produced by bacterium Streptomyces

albulus.

• 25-30 (35) lysine monomers.

• Mw 3000-4000Da

Food Applications of EPL

• 1. antimicrobial agent • against both Gram(+) and Gram(-) bacteria

• because of its positive charge

• GRAS (2004), in cooked rice or sushi rice

• 2. dietary agent

• inhibit pancreatic lipase

• suppress dietary fat absorption

ε-Poly(lysine)-Based Micelles

• EPL + octenyl succinic anhydride (OSA)

(J. Agr. Food Chem. 2010, 58, 1290-1295)

Physical Properties

Samples Tg (oC)

EPL 133.1

OSA-g-

EPL6.2 126.1

OSA-g-

EPL8.5 94.0

OSA-g-

EPL12.4 78.9

OSA-g-

EPL20.5 60.6

Micelle Structure

Q (A-1)

10-2 10-1

I(Q

) a

.u.

10-5

10-4

10-3

10-2

OSA103

OSA106

OSA110

OSA103 fit

OSA106 fit

OSA110 fit

Pair distribution function Small-angle x-ray scattering profiles

Antimicrobial vs.

Cytotoxicity

Minimum Inhibition Concentration

(MIC)=12.5 (μg/mL)

Cytotoxicity in Hep G2

cells was found for OSA-g-EPL

between 300-600 μg/mL.

Three loading methods to compare the

encapsulation capacity

Encapsulated curcumin showed

increased cellular antioxidant activity

**: P<0.01

Conclusions of micellar encapsulation

experiments

• Curcumin was able to be solubilized in micelle

solution, exampled by micelles formed by

modified starch and newly synthesized modified

epsilon polylysine.

• Upon encapsulation, in vitro bioactivity of

curcumin was increased, suggesting that solubility

limited the cellular absorption of curcumin.