Microcapsule Design Considerations and...

Microcapsule Design Considerations and Characterisation

CSIRO FOOD AND NUTRITION

Luz Sanguansri & MaryAnn Augustin

Short Course on Micro- and Nano-encapsulation of Functional Ingredients in Food Products World Congress on Oils & Fats and 31st Lectureship Series 31st Oct – 4th November 2015, Rosario, Argentina

Outline

• Do you need microencapsulation?

• Why is microencapsulation required?

• What are important factors to consider?

• How is it done?

• Testing and characterisation of microcapsules

• Summary

2 | Sanguansri & Augustin | CSIRO

Do you need microencapsulation?

Bioactives identified for health

Microencapsulation required

Microencapsulation not required

Formulate directly into Food

Microencapsulated ingredient

Application and Formulation into

food

Is the ingredient stable in current form?

NO YES


Why is microencapsulation required?

• For stabilisation of bioactive

• Many are unstable once they are isolated

• Protection required throughout shelf-life

– Under storage conditions that ingredient is exposed to

• For enabling delivery into food

• Masking taste – allowing addition without compromising sensory appeal

• Preventing undesirable interactions with other food components

– Stability during food processing

– Stability in final food product

• For enhancing bioavailability of bioactive

• Target delivery to site to exert desired physiological function

– Depends on intended health benefit


5 |

Nutraceutical Potential benefits of encapsulation

Omega-3 fatty acids Protection from oxidation | Powder format (convenience) | Taste masking

Controlled release | Enables incorporation in aqueous based food and

beverages

Probiotics Improve viability during storage | Protection in food product

Protection from stomach acids and bile

Phenolic compounds and

polyphenols

Taste masking | Improved solubility | Improved bioavailability

Facilitates incorporation into food products

Lipophilic phytochemicals

(e.g. carotenoids,

tocopherols)

Protection from oxidation | Powder format (convenience) | Controlled release

Enables incorporation in aqueous based food and beverages

Bioactive peptides Masking bitterness and astringency | Controlled delivery

Protection from acid environments

Minerals Taste masking | Avoidance of undesirable interactions (e.g. Fe-catalysed fat

oxidation, Ca-induced protein precipitation)

Vitamins Protection against degradation | Taste masking | Controlled release

Ease of incorporation of fat soluble vitamins in aqueous based foods and

beverages

Examples of potential benefits of encapsulation

Sanguansri & Augustin | CSIRO

What are the important factors to consider?

• Active ingredient (core) - properties

• Target application - format

• Processing conditions & capabilities

• Release mechanism

• Particle size

• Volume

• Load required

• Storage stability

• Other (legal, natural, etc)

• COST


Sample questionnaire (1 of 2)

7 |

Product:

□ Core/bioactive - provided MSDS, specification and certificate of analysis

o Stability to temperature, pH, shear, oxygen, moisture, light, UV

o Format: liquid, powder, crystal, etc,

o Purity or amount of active if it is in a carrier (specify carrier)

o Shelf-life : Storage temperature, months

□ Microencapsulated product description:

o General description

o Product format: (e.g. powder or liquid)

o Payload (active / oil content)

o Particle size

o Special requirements:

Stability of microencapsulated product:

□ Acceptable % loss during shelf life (commercial ingredients)

□ Packaging and storage conditions (temperature, months)

□ Expected shelf-life

□ Other requirements

Application of microcapsules in final application:

□ Food product / other (specify)

□ Process requirements in final application

□ Bioactive fortification level in final application per serving or per 100g

□ Packaging and storage condition of final product

□ Stability and shelf life of final product


Sample questionnaire (2 of 2)

8 |

Reason for encapsulation

□ convert liquid to powder / solid

□ protect from degradation

□ mask taste, odour

□ stable under high shear

□ stable under compression

□ provide moisture barrier

□ controlled release

□ increase solubility

□ increase bioavailability

Other information (if available)

□ Do you manufacture the bioactive ingredient to be encapsulated or supplied by a third party?

□ Does your company plan to manufacture the microencapsulated product, or by a third party?

□ Is there an existing commercial product to match or improve? (appearance, flow properties,

composition, stability, etc)

□ What is the characteristic of the current product to match or improve?

□ Do you need a sample for your own evaluation? (how much sample required)

□ What would success look like during initial evaluation phase? (e.g. 5-10% loss at accelerated oxygen

atmosphere, room temperature for 20 days)

□ What is your timeline to commercialization (if successful)?


Elements of Microcapsule Design

Core Material Formulation Process ME product

Select/

identify

Existing/

New

Design formulations Conventional/ emerging

Develop specifications & applications

Characterise – physical & chemical

Modification / Synthesis

Study of interfacial behaviour

Maintain/ develop processing capability


Stability & solubility


Study of ingredient interactions

Assess effects of processing

Stability of ME product – e.g. heat, pH, moisture

Stability of materials

Triggers for release & stability of formulation

Assess efficiency /consistency of process

Suitability for incorporation into foods/ target delivery

Work with supplier / user

Establish cost / availability

Compare with competitors / existing products

Align with industry capability

Align with end-user, Work with nutritionists

AC

TIV

ITY

SC

IEN

CE

MA

RK

ET

Chemistry, Material Science

& Engineering

Formulation science & Chemistry

Engineering and Processing

Chemistry, Biochemistry, Food

technology, Nutrition

Chemistry, Microbiology

CA

PAB

ILIT

Y

CSIRO©


Generic steps to designing microcapsules

Design Delivery

System Formulation

& Manufacture

Characterise

Release

Properties

Food Application

& Delivery to the

Gastrointestinal

Tract

•Identify the core

•Choose encapsulant material

•Define microcapsule requirements

•Design formulation

•Prepare microcapsule

•Analyse to quantify the core

•Test microcapsule properties

•Test stability and sensory properties

•Test in-vitro release properties

•Test stability in food application

- stability during processing

- stability during storage

•Test sensory properties in final food

•Test in-vitro release

•Test in-vivo & bioavailability

Functional Food Product

1

2

3

refo

rmu

late


Testing & Characterisation of Microcapsules

o Considerations in deciding the methodology

o Physical characterisation and analysis of emulsions

o Characterisation of powder microcapsules

o Characterisation of microcapsules to monitor stability of the core from degradation

o Release properties of microcapsules

Introduction…

• Choice of formulation, processing and format of the microcapsule are dependent on the active and final application...

• there are generic analysis that are common

• the task of selecting the most appropriate methodology is often dependent on final application

• Diversity of applications make it difficult to cover all aspects in detail

• Will consider the task conceptually and provide examples

Encapsulant Selection (formulation)

Method of Processing (production)

Method of Testing (characterisation)


Main consideration before and during analysis - possible effect of matrix / encapsulant

• Must be sure that active is released

so it can be measured • Some matrices are hard to release the active encapsulated

• Incomplete release can under estimate amount of active

• Choice of solvent for release is important

• Make sure wall/matrix materials do not interfere with analysis of the active • For probiotics: complete release and dispersion of cells is important to

count individual cells vs. group of cells


Other consideration during analysis

For lipid extraction:

• Choice of solvent – matrix dependent

• Time duration

• Separation method – centrifuge, filter, stand

For active quantification:

• Active extraction, separation and sample preparation

• Analytical method and instrumentation

For probiotics viability:

• Rehydration and growth media

• Enumeration methodology


Encapsulation Efficiency (ME)

• For lipid/oil encapsulation – by oil extraction

• ME = [(total oil – surface oil) / total oil ] x 100%

• For active encapsulation – by GC or HPLC

• ME = [amount of active after encapsulation / amount of active added in formulation] x 100%

• For probiotic encapsulation – total plate count

• ME = [viability (cfu/g) after encapsulation / viability (cfu/g) added in formulation ] x 100%


Physical properties

Emulsion • Particle size

• Viscosity

• Oil-water interface

Powders

• Moisture content

• Water activity

• Particle size

• Bulk density

• Flowability

• Dispersibility & solubility

• Morphology and structure


Storage Stability

Physical stability • Emulsions - separation

• Powders - caking

Chemical stability • Lipid oxidation

• Active degradation

Stability are influenced by storage conditions • Packaging

• Environment – temperature, humidity, oxygen, light


Stability to stresses during processing & storage

Simulate processing stresses • Temperature stresses – UHT, retort

• High shear stresses – homogenisation

• High shear-temperature combination – extrusion

Simulate the environment in final food application • Consider the moisture and water activity environment in final product

application

– dry, moist or liquid food

– high-, low-, neutral- pH

Simulate the storage and market environment – Consider the temperature and relative humidity of where the

product will be sold


In-vitro release testing

Simulate the release “event” • Time

• Process

• Environment

– Water

– Simulated gastric fluid

– Simulated intestinal fluid

– Sequential exposure to simulated fluids


Characterisation of Emulsions

Examples

Free fat: used to optimise formulation for encapsulation of lipids

0

2

4

6

8

10

12

14

16

WPI WPC-80 a-lac NaCas SPI

free fat

0

1

2

3

4

5

6

glucose sucrose lactose DGS(DE24)

free fat

0

2

4

6

8

10

12

14

WPC

:suc(

40%

fat)

Nca

s:su

c(40

%fa

t)

WPC

:suc(

60%

fat)

Nca

s:su

c(60

%fa

t)

30% TS

40% TS

0

5

10

15

20

25

30

Ncas:lac(40%fat) WPI:lac-suc(60%fat)

1:1

1:2

Effect of protein type

Effect of protein: carbohydrate ratio

Effect of carbohydrate type

Effect of total solids


Particle size: Used to measure quality of emulsion from reconstituted microcapsules

Particle size distributions of reconstituted microcapsules at 0 (A), 1 (B), 2 (C) and 3 (D) months storage under ambient conditions

Polavarapu et al., 2010, Food Chem 22 | Sanguansri & Augustin | CSIRO

Turbiscan: used to monitor emulsion stability

Change in backscattering at various position along the sample measurement path indicates instability…

-Coalescence

-Flocculation

-Creaming

-Sedimentation

Stable emulsion Unstable emulsion

d(0.5) = 1.1µm

d(0.5) = 3.9µm

d(0.5) = 6.4µm

Back Scattering

0mm 20mm 40mm 60mm

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0:00

0:01

0:03

0:04

0:06

0:08

0:10

Back Scattering

0mm 20mm 40mm 60mm

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0:00

0:01

0:03

0:04

0:07

0:09

Back Scattering

0mm 20mm 40mm 60mm

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0:00

0:01

0:02

0:05

0:10


Light microscopy and free fat - used to characterise emulsion properties

0.1% casein

0.25% casein

1.0% casein

100µm

100µm

100µm

Day et al, 2007, Food Chem 24 | Sanguansri & Augustin | CSIRO

CLSM used to visualise emulsion structure and quality (encapsulation efficiency)

Free oil visible(Protein and un-processed starch)

Stable emulsion(Protein and pre-processed starch)

Unencapsulated oil visible(OSA starch)

Aggregated structure(Protein, sugar and pre-processed starch)

Very fine structure(Protein and sugars)

Fat stained Nile Red (RED); Protein stained FITC (GREEN)

50% oil loading powders


CLSM used to visualise change in emulsion structure after addition of polysaccharide

Primary emulsion + pectin (pH7) Stained FITC(GREEN) for protein,

Nile Red for fat

Primary emulsion (Protein sugars) Stained FITC for protein,

Nile Red for fat

Primary emulsion + pectin (pH7) Stained Congo Red for polysaccharide


Characterisation of Powder Microcapsules

Examples

DVS to measure moisture sorption properties - relates to powder flow characteristics (hygroscopicity)

DVS Change In Mass (dry) Plot at 25C

0

2

4

6

8

10

12

14

16

18

0 100 200 300 400 500 600 700 800 900 1000

Time/mins

Ch

an

ge

In

Ma

ss

(%

) -

Dry

0

10

20

30

40

50

60

70

80

90

100

Ta

rge

t R

H (

%)

WPI/Hylon(1:1)/50%oil Cas/Hylon(1:1)/50%oil Target RH

Casein and whey protein have similar MW, but different structures and inherent water binding properties

0

10

20

30

40

50

60

70

80

0 200 400 600 800 1000 1200 1400 1600

Time/mins

Rela

tive H

um

idit

y i

n %

-2

0

2

4

6

8

10

12

14

Mass C

han

ge i

n %

© Surface Measurement Systems Ltd UK 1996-98DVS - The Sorption Solution

Influence of matrix on water uptake


SEM used to visualise morphology & structure of powder microcapsules

Dried coacervates Spray dried formulation with starch coating

Spray dried emulsion

internal & external structure



Sanguansri & Augustin, 2010, Wiley Blackwell 29 | Sanguansri & Augustin | CSIRO

Compression testing for mechanical strength of microcapsules

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)

Fo

rce (

kN

)

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)

Fo

rce (

kN

)

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)

Fo

rce (

kN

)

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)

Fo

rce (

kN

)

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)

Fo

rce (

kN

)

0

1

2

3

4

5

6

0 5 10 15

Distance (mm)F

orc

e (

kN

)

Formulation 1 (F1) Formulation 3 (F3)

F1 + polysaccharide

Formulation 2 (F2)

F2 + polysaccharide F3 + polysaccharide

Addition of polysaccharide improves mechanical strength of particles


CLSM used to visualise powder structure - location of fat and protein in the matrix

Good Powder structure (spherical particles – oil inside particles)

Poor Powder structure (irregular particles - with free oil)

Pro

tein

sta

ine

d w

ith

A

crid

ine

Ora

nge

Pro

tein

sta

ine

d w

ith

A

crid

ine

Ora

nge

Fat staine

d w

ith N

ile

Re

dFat stain

ed

with

Nile

R

ed


CLSM used to visualise the location of cells within the microcapsules

Protein-carbohydrate-lipid Emulsion matrix

Protein matrix Resistant Starch matrix


CLSM used to visualise probiotic cells - intact vs compromised cell membrane

Live and dead cells Live cells Mostly dead cells

SYTO9 stain (LIVE, GREEN) ; Propidium Iodide stain (DEAD, RED)


Stability of the Core During Storage

MUST reproduce commercial storage conditions oTemperature

o Relative humidity

o Packaging

Oxipress to test resistance of sample to oxidation under accelerated storage condition

Fish oils

Encapsulated omega-3 oil powders

“induction period (IP)” - indicates resistance of sample to oxidation

“Slope” – indicates of how fast is the reaction during IP

IP


Peroxide value for fat oxidation

Bao et al 2011, JFS

Stability of n-3 microcapsules stored at 30°C (in NaCas transglutaminase matrix)


EPA

IS

DHA

5 0

EPA

IS

DHA

Acid methylation: triglyceride + free fatty acids

Base methylation: glyceride bound fatty acids

GC Analysis: - for quantification of omega-3 EPA and DHA


HPLC (ELSD) or Iatroscan for Lipid class analysis e.g. mono-, di-, triglycerides, and phospolipids)

500x103

400

300

200

100

0

EL

SD

res

po

ns

e (

mV

)

2520151050

Elution Time (min)

Free fatty acids Diglycerides

Triglycerides

Monoglycerides

Lipid class analysis of digested triglyceride oil (HPLC)

mv

minutes

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

0

5

10

15

20

25

30

35

0.132 13173

0.464 29246

Lipid class analysis of phospholipid rich marine oil (Iatroscan)


Storage Stability of omega-3 powder microcapsules - sensory, headspace and fatty acid analysis

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

0 m

onth

3 m

onths

6 m

onths

9 m

onths

12 m

onths

15 m

onths

18 m

onths

24 m

onths

Storage Time

Pro

pan

al

Co

nte

nt

(ug

/g)

MicroMAX (50% oil) 25°C MicroMAX (50% oil) 35°C

Threshold for detectible rancidity

0.0

3.0

6.0

9.0

12.0

15.0

0 m

onth

3 m

onths

6 m

onths

9 m

onths

12 m

onths

15 m

onths

18 m

onths

24 m

onths

Storage Time

Ove

rall

Qu

ali

ty


0.0

3.0

6.0

9.0

12.0

15.0

0 m

onth

3 m

onths

6 m

onths

9 m

onths

12 m

onths

15 m

onths

18 m

onths

24 m

onths

Storage Time

Ran

cid

Fla

vo

ur

Inte

nsit

y


0.0

20.0

40.0

60.0

80.0

100.0

0 m

onth

3 m

onths

6 m

onths

9 m

onths

12 m

onths

15 m

onths

18 m

onths

24 m

onths

Storage time (months)

% o

meg

a-3

an

d t

rig

lycerid

e

co

ncen

trati

on

% DHA % EPA % Triglyceride

Sensory Analysis – taste panel

Fatty acid analysis – GC Lipid class (triglyceride) - Iatroscan

Sensory Analysis – taste panel

Head space analysis - GC


HPLC analysis to monitor stability of microencapsulated tocopherols

Less than 10% loss over 12 months storage at 25°C

(vacuum packed in aluminium foil sachets)


HPLC: analysis of resveratrol in microcapsules GC: analysis of tributyrin in microcapsules

18 months storage stability of Tributyrin in microencapsulated powder

containing a cocktail of bioactives stored at 25°C

0.0

10.0

20.0

30.0

40.0

50.0

0M 3M 9M 18M

mg

/ g

po

wd

er

0

20

40

60

80

100

-3 0 3 6 9 12 15 18Storage time (M, Axis for %R)

% R

em

ain

ing

MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res)MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res)

18 months storage stability of Resveratrol in microencapsulated powder

containing a cocktail of bioactives stored at 25°C

0.000

0.050

0.100

0.150

0.200

0.250

0M 3M 6M 9M 18M

mg

/ g

po

wd

er

0

20

40

60

80

100

-3 0 3 6 9 12 15 18

Storage time (M, Axis for %R)

% R

em

ain

ing

MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res)MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res)

Shelf stability of resveratrol in a mixture of bioactive

Greater than 95% remaining after 18 months storage

at 25°C

Shelf stability of tributyrin in a mixture of bioactive

85-90% remaining after 18 months storage at

25°C

Bioactive cocktail combination: Omega-3 DHA & EPA, resveratrol, Tributyrin


Stability of microencapsulated probiotics during storage at high Aw

0.00000001

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

100

0 2 4 6

Tim e (w e e ks)

Pe

rce

nt

su

rviv

al

(Lo

g

sc

ale

)1

0

Encapsulated

probiotics

Non-encapsulated

probiotics

10 μm

10 μm

Storage Stability (25°C, 50% RH)

Crittenden et al. 2006, AEM, 72(3), 2280-2282 42 | Sanguansri & Augustin | CSIRO

Release properties of Microcapsules

Test conditions MUST be under

representative conditions!

Survival of microencapsulated probiotics during in-vitro digestion

0.001

0.01

0.1

1

10

100

Non-encapsulated

probiotic

Encapsulated

probiotic

Perc

en

t su

rviv

al (L

og

scale

)

In-vitro survival: Encapsulated vs. non-encapsulated

A B

In gastric fluid In intestinal fluid

In-vitro Release

In vitro model: US Pharmacopeia SGF 1h pH 1.2, trypsin SIF 3h pH 6.8, pancreatin

USP method: incubation at 37°C in SGF (pH 1.2, 2 hrs) then in SIF plus bile salts and calcium (pH 6.8, 3 hrs)

Crittenden et al. 2006, AEM, 72(3), 2280-2282 44 | Sanguansri & Augustin | CSIRO

pH stat used to characterise digestibility of powder microcapsules in-vitro

In-vitro release during incubation at 37°C in simulated intestinal fluid plus bile salts and calcium (pH 6.8) for 3 hrs – USP method

0

50

100

150

200

250

300

0 30 60 90 120 150 180

SIF Time (min)

Ac

id R

ele

as

ed

(u

Mo

l)

Cas-oligo DGS MRP

SPI Pectin MRP

WPI Hylon MRP First

Cas Hylon MRP First

Cas Hylon MRP Last

Intralipid

CSIRO, unpublished data 45 | Sanguansri & Augustin | CSIRO

In-vitro release of Omega-3 from microcapsules

Bao et al 2011, JFS

Encapsulant: Cross‐Linked NaCas and Transglutaminase

In-vitro release during incubation at 37°C in pepsin solution (2mg/mL citric acid, pH 2), filtered , dried, and weight of filter paper recorded


13C NMR used to characterise digestibility of microcapsules in-vitro (mobile components)

powder

In water

SGF+SIF

(no bile/Ca)

SGF

oil oil oil oil Encapsulant Encapsulant

Burgar et al. 2009, Food Biophysics, 4, 32-41

Protein-carbohydrate blend formulation

Non-MicroMAX

Heated protein-carbohydrate with RS formulation

MicroMAX®


CLSM used to visualise structure of microcapsules during in-vitro digestion

Protein stained FITC (GREEN); Fat stained Nile Red (RED)

Before digestion After SGF 2h, pH 1.2 After SIF 5h, pH 6.8

No

n-M

icro

MA

X

Mic

roM

AX

Chung et al. 2011, Food Chem, 124, 1480-1489 48 | Sanguansri & Augustin | CSIRO

Light Microscopy: used to visualise change in structure of food matrix during in-vitro digestion

SGF 2h, pH 1.2

SIF 5h, pH 6.8

Powder in Yogurt Powder in Cereal Bar

Powder in Orange Juice

Neat Powder

Shen et al. 2011, JAFC, 59, 8442-8449 49 | Sanguansri & Augustin | CSIRO

Effect of food matrix – release of omega-3 fatty acids

0

400

800

1200

1600

2000

2400

2800

3200

Fish oil capsule Orange juice Yogurt Cereal bar

Re

co

ve

red

EP

A a

nd

DH

A (

mic

ro g

ram

)

EPA

DHA

0

10

20

30

40

50

60

70

80

90

control orange juice yogurt cereal bar

% O

me

ga

-3 f

att

y a

cid

s

Digested Samples

B

EPA DHA

The food matrix can influence the release of long chain omega-3 fatty acids during in-vitro digestion and in-vivo trial

In-vitro digestion in SGF (2hrs, pH 1.2) and SIF

(5hrs, pH 6.8)

In-vivo trials in ileostomy patients

(<2% of dose recovered from ileal digesta)

Shen et al, CSIRO, Unpublished data 50 | Sanguansri & Augustin | CSIRO

Points to remember during testing…

• Choice of method for testing and characterisation of microcapsule is dependent on core, encapsulant material, format and application

• Must be sure that active is released so it can be measured

• Must ensure that the encapsulant materials do not interfere with analysis

• Storage Stability – MUST reproduce storage conditions e.g. temperature, relative humidity, packaging

• Release – test conditions MUST be under representative conditions


Overall Summary

Microcapsule design

• Important to ask the right questions at the start

• Define the specification/requirements of the microcapsule

• Choose the correct method to test/characterise the microcapsule

Ingredient / Product requirement

• Stability – important for quality, shelf life

• Taste – should not be compromised

• Bioavailability – important for health and wellness products

Commercial / Regulatory

• Cost – still remains a major consideration for successful commercialisation

• Regulatory standards – market jurisdiction


Luz Sanguansri Research Team Leader

t +61 3 9731 3228 e [email protected] w www.csiro.au

Mary Ann Augustin Research Group Leader

t +61 3 9731 3486 e [email protected] w www.csiro.au

CSIRO FOOD AND NUTRITION

Thank you

Microcapsule Design Considerations and...

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