emerging technologies & case studies - asaga · •the supercritical temp and pressure for co 2...

Emerging Technologies & Case Studies

Luz Sanguansri & MaryAnn Augustin

CSIRO FOOD AND NUTRITION

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

• Trends in Microencapsulation • Publications, patents, market

• Emerging Technologies • Clear Beverages

• New Processes

• Case Studies - Bioactive delivery

• Omega-3 Oils

• Bioactive Cocktail (Fish oil, Resveratrol, Tributryin)

• Probiotics

• Conclusion

Emerging Technologies | Augustin & Sanguansri 2 |

Patents in Encapsulation – Food & Beverage

Sobel et al. Microencapsulation in the Food Industry, 2014, 3-12


Trends in Microencapsulation – Papers published

Coacervation

Spray drying

Nanoencapsulation

Spinning disk

RESS

Liposome entrapment

Spray cooling

Fluidized bed

Extrusion

Sobel et al. Microencapsulation in the Food Industry, 2014, 3-12


Market & trends - Food Encapsulation

Source: *http://www.marketsandmarkets.com/Market-Reports/food-encapsulation-advanced-technologies-and-global-market-68.html ** Global Business Insights – Innovations in delivery methods for nutraceutical food and drinks, 2011

Global Encapsulation Market*

$39.5 billion in value by 2020

CAGR of 6.1% from 2015

Target Groups/Markets:

•Infant, functional and health food segments

•Ageing population

•Foods with disease prevention benefits.

Concerns – Nanoencapsulation:

“Whilst this is in part due to potential and unknown toxicity relating to nanoparticles, it is also the case that nanoencapsulation is rarely the best solution”**

Food encapsulation market size by

Core / active ingredients*

Emerging Technologies | Augustin & Sanguansri

Microencapsulation for Clear Beverages


Food Grade Nano-emulsions formed by spontaneous emulsification and gelation

Nanoemulsion Production

• Addition of organic phase (oil + surfactant) to an aqueous phase

• Optical clarity increased when spontaneous emulsification was performed at higher temperature

Formation of Translucent filled hydrogels

• Nanoemulsion incorporation into a model gelatin system and a gelatin dessert had little effect on their rheological characteristics

• Slight changes in their optical properties (but still gave translucent hydrogels

Lipophilic bioactives can be incorporated into functional food gels using this method

Komaiko and McClements (2015) Food Hydrocolloids, 46, 67-75


Soybean β-Conglycinin Nanoparticles for delivery of hydrophobic nutraceuticals


• Nanoparticles were prepared by dissolving dry VitD3 into EtOH and adding to β-CG solution

• VD-β-CG nanoparticles had mean diameters in the 50–400 nm range

Function

• β-CG also protected Vit D from several forms of degradation

• Shelf life test - nanoparticles retained 70 % and 90 % of their vitamin content at pH 6.8 and 2.5, respectively, whereas the protein-free controls retained only about 13–22 %

Lipophilic bioactives can be incorporated into functional food gels using this method

Levinson et al (2014) Food Biophysics 9, 332–340

Vit D3 (Protein-

free) in pH 6.8

buffer

Vit D3 in protein

nanoparticles in

pH6.8 buffer


Encapsulation of eugenol using Maillard-type conjugates to form transparent and heat stable dispersions


• Eugenol was encapsulated with whey protein isolate – maltodextrin conjugates formed by Maillard reaction and spray dried

• Nanoemulsions made with Maillard conjugates were more transparent than those made with non-conjugated mixtures.

Functionality

• Encapsulated essential oil incorporation into beverages at pH 3 and pH 7

Essential oils can be incorporated into clear beverages

Shah et al (2012) LWT Food Sci & Tech 49, 139–148

Eugenol emulsions formulated with

1WPI:2 MD40 at various pH (3, 5, 7)

Heating – 85C/15 min


Emerging Processes


Emerging Processes

Size Reduction / Emulsification

• Conventional – Homogenisation, Milling (superfine milling)

• Newer – Microfluidisation, Membrane emulsification, Ultrasonication

Atomisation

• Conventional – Spray drying

• New – Electrospraying

Gas Expansion

• Supercritical fluid-based processes

• Extrusion Porosification


Equipment for production of nanoemulsions

12 |

McClements & Rao (2011) Crit Rev Food Sci & Nutr, 51, 285-330


Electro-spraying

• Electrospraying is a method of liquid atomisation by electrical forces

• Advantages:

• Smaller droplet size (<1 micron) than conventional atomisers

• Narrow size distribution

• Droplets are charged – self dispersing, absence of agglomeration and coagulation


Supercritical CO2 assisted microencapsulation

14 |

• Use compressed CO2 in liquid or supercritical state as the solvent • Steps:

• polymer is exposed to supercritical CO2 for a while;

• the solution of additives in CO2 is introduced and the solute is transferred from CO2 to polymer;

• CO2 is released and the solute is trapped in the polymer material • High volatility of CO2 allows it to be easily separated from polymeric materials by

lowering the pressure • The supercritical temp and pressure for CO2 to be in fluid state is low e.g. 31°C and 74

bar respectively


Eric Keven Silva, M. Angela A. Meireles*

Food and Public Health 2014, 4(5): 247-258

Extrusion Porosification Technology (EPT)


• EPT™ uses Continuous twin-screw technology

• Useful in texturing powders to provide them with new functions.

• The twin-screw extruder is configured to continuously process highly viscous and heat-sensitive products

• Uses mechanical action adapted to the function (mixing, limited shearing, controlled residency time) and precise temperature control.

www.Clextral .com

http://www.clextral.com/technologies-and-lines/technologies-et-procedes/twin-screw-extrusion-technology/




http://www.clextral.com/technologies-and-lines/equipment/twin-screw-extruders/



Sol-Gel Encapsulation

• Sol-gel encapsulation allows trapping lipophilic components inside the spherical shell of amorphous silicon dioxide

• Steps:

• prepare an o/w emulsion with an active solubilised in the silicon phase e.g. tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS)

• Hydrolyse the silicon droplets then condensation of the hydrolysed species to silica occurs at the oil-water interface to led to formation of the hard silica shell.

16 | Emerging Technologies | Augustin & Sanguansri

Case Studies - Bioactive delivery


Bioactives of Interest Bioactive Examples Sources

Omega-3 fatty acids α-linolenic acid (ALA) Flax, perilla, chia

Eicosapentaenoic acid (EPA) &

Dodecahexaenoic acid (DHA)

Fish oil, marine algae, krill oil

Probiotics Lactobacilli, Bifidobacterium Cultured microorganisms

Prebiotics Inulin Chicory root, Jerusalem artichoke, jicama

β-glucan Barley, oats

Carotenoids β-carotene Carrots, sweet potato, palm oil, algae

Lycopene Tomato, water melon, red grapefruit

Lutein and zeaxanthin Nasturium (yellow flowers), kale, spinach

Phenolic compounds &

Polyphenols

Resveratrol Japanese knotweed, wine

Curcumin Tumeric

Flavonoid (quercetin & rutin) Onions

Flavonoids (hesperitin) Orange juice

Catechins & epicatechins Cocoa, chocolate, tea


Bioactives and their health benefits Bioactive Examples Potential health benefits

Prebiotics Inulin, oligosaccharides Promoting gut health; modulation of gut microflora

Probiotics Lactobacilli,

Bifidobacterium,

Improving gut health, immune modulation

Phytochemicals Beta-carotene, lycopene,

flavonoids,

proanthocyanidins,

polyphenols, allicin

Reducing risk of cardiovascular disease, cancer,

diabetes, and age-related degenerative diseases

Long chain omega-3 fatty

acids

Dodecahexaenoic acid

(DHA), eicosapentaenoic

acid (EPA)

Promoting cardiovascular health

Bioactive peptides Milk-derived peptides Reducing blood pressure

Carotenoids Beta-carotene, lycopene,

lutein, zeaxanthin,

astaxanthin

Reducing risk of eye diseases and certain cancers

Herbs and spices Essential oils, various

herbal preparations

Range of health benefits


- Omega-3 oils

Milk-protein based microencapsulated bioactives - Stability and Bio-availability (MicroMAX Technology)


MicroMAX: Our Technology Platform

An innovative microencapsulation

“technology platform” for

protection and delivery of

bioactives into functional food

• Using natural food grade materials

• Using simple chemistry

• No additives

• Utilising standard food processing

equipment


Production Of Omega-3 powders using Food Materials

ENCAPSULANT MATERIAL + FISH OIL

STABILIZED LIQUID EMULSION

SPRAY-DRIED EMULSION

Emulsification

Spray drying

Coating material & encapsulation technique influences the properties of the microcapsules


http://images.google.com.au/imgres?imgurl=http://www.itogha.com/uploads/kundefiler/1. Oil4Life Liquid.jpg&imgrefurl=http://www.itogha.com/index.php?sideID=79&usg=__Ow58qDF_3EAeWi4i39A0yBgR7bo=&h=841&w=561&sz=216&hl=en&start=22&um=1&itbs=1&tbnid=tx3N_3m0q4qp3M:&tbnh=145&tbnw=97&prev=/images?q=liquid+oil&start=20&um=1&hl=en&sa=N&rls=com.microsoft:en-US&ndsp=20&tbs=isch:1

Shelf-life of fish oil powders: MicroMAX®(50% oil) versus Non-MicroMAX®(25% oil)

MicroMAX® Powder compared to Non-MicroMAX Powder

Doubled the oil loading

Doubled the shelf life

0.0

3.0

6.0

9.0

12.0

15.0

0 mont

h

3 mont

hs

6 mont

hs

9 mont

hs

12 m

onth

s

15 m

onth

s

18 m

onth

s

24 m

onth

s

Storage Tim e (m onths )

Ra

ncid

Fla

vo

ur

Driphorm-50 25°C Driphorm-50 35°C

Driphorm-HiDHA 25°C Driphorm-HiDHA 35°C

Detectible

rancidity

50% oil powder (MicroMAX)

25% oil powder (Protein-CHO Blend)

MicroMAX®-50%oil(25°C)

Non-MicroMAX®-25%oil(25°C)



Detectible

rancidity

50% oil powder (MicroMAX)

25% oil powder (Protein-CHO Blend)




Non-MicroMAX®-25%oil(35°C) Clover Corp

– CSIRO


MicroMAX Application in dairy products

Omega-3 fortification using MicroMAX powder (50% oil) or MicroMAX UHT

emulsion (25% oil) is better than using unencapsulated oil

CSIRO: Sharma, R. et al AJDT, 2003

Dosage: 60mg DHA+EPA per 110g serving (yoghurt) or per 20g serving (processed cheese /dip)


In-vitro testing (Microcapsules & Food Matrices containing Microcapsules)

Simulated Gastric Fluid (SGF)

(NaCl/ HCl /pepsin)

pH 1.2, 37°C for 2 hr

Simulated intestinal fluid (SIF)

(phosphate, pancreatin, bile salt, Ca)

pH6.8, 37°C for 3 hr

Analysis

% Released fat (solvent extraction)

% Lipolysis (Free fatty acids)

Imaging

Low pH

Gastric enzymes

Bile & Intestinal

enzymes

Gut

microflora

Prior to

digestion SGF SGF&SIF



Human study: % change in EPA & DHA in plasma of human volunteers over 48 hr with single dose of omega-3 (~1.0 g DHA+EPA) with flavoured milk

0 1 0 2 0 3 0 4 0 5 0

0 .0

0 .2

0 .4

0 .6

T im e (h )

% D

elt

a 2

0:5

n-3

(E

PA

)

***

**

A

0 1 0 2 0 3 0 4 0 5 0

0 .0

0 .2

0 .4

T im e (h )%

De

lta

22

:6n

-3 (

DH

A)

*

B

0 1 0 2 0 3 0 4 0 5 0

0 .0

0 .2

0 .4

0 .6

0 .8

T im e (h )

% D

elt

a t

ota

ln

-3 F

A

*

*

C

Change (delta) from baseline in the fatty acids as % of the total fatty acid pool in

plasma (n=15).

Fish oil gel capsules taken with a flavoured milk (o); Flavoured milk containing fish oil microcapsules

(MicroMAX1) (□)

Flavoured milk containing fish oil microcapsules (MicroMAX2) ()

Some minor differences in rate of change between samples but

no significant differences at 24 and 48 hr Sanguansri et al 2015, Brit J Nutr 113, 822-

831,


Human study – short term time course: omega-3 and omega-6 fatty acids as % of total fatty acid pool in erythrocyte membranes after daily dose (1.0 g EPA +DHA)

Change (delta) from baseline in the fatty acids as % of the total fatty acid pool in

erythrocyte membranes (n=47).

Fish oil gel capsules taken with a flavoured milk (o); Flavoured milk containing fish oil microcapsules

(MicroMAX1) (□)

Flavoured milk containing fish oil microcapsules (MicroMAX2) ()

Bioequivalence of fish oil microcapsules (MicroMAX) and gelatine

fish oil capsules when taken with flavoured milk

0 2 4

-2 .0

-1 .5

-1 .0

-0 .5

0 .0

0 .5

1 .0

1 .5

2 .0

n -3 F A

n -6 F A

A

T im e (w e e k s )

% D

elt

a F

att

y A

cid

s

a

b

b

b

b

0 2 4

0 .1 5 0

0 .1 7 5

0 .2 0 0

0 .2 2 5

0 .2 5 0

0 .2 7 5

0 .3 0 0

B

T im e (w e e k s )

n-3

/n-6

FA

ra

tio

a

b

b

Sanguansri et al 2015, Brit J Nutr 113, 822-831,

- Bioactive Cocktail (Fish oil, Resveratrol, Tributyrin)



Formulation of a Bioactive Cocktail ENCAPSULANT MATERIAL + BIOACTIVES

STABILIZED LIQUID EMULSION

Emulsification

Protein

Carbohydrates

Cocoa butter

Fish oil

Tributyrin

Resveratrol POWDER

Microencapsulation Efficiency: 96-99% (for all bioactives)

Milk protein-based microencapsulated bioactives |MA Augustin

BIOACTIVES

ENCAPSULANT


http://images.google.com.au/imgres?imgurl=http://www.itogha.com/uploads/kundefiler/1. Oil4Life Liquid.jpg&imgrefurl=http://www.itogha.com/index.php?sideID=79&usg=__Ow58qDF_3EAeWi4i39A0yBgR7bo=&h=841&w=561&sz=216&hl=en&start=22&um=1&itbs=1&tbnid=tx3N_3m0q4qp3M:&tbnh=145&tbnw=97&prev=/images?q=liquid+oil&start=20&um=1&hl=en&sa=N&rls=com.microsoft:en-US&ndsp=20&tbs=isch:1

http://upload.wikimedia.org/wikipedia/commons/1/17/Resveratrol.svg

Microencapsulated Bioactive Mixture containing Fish Oil, Tributyrin and Resveratrol

Bulk Phase

Core - oil

Protein-resveratrol complex Resveratrol

Resveratrol

Resveratrol

Resveratrol


http://upload.wikimedia.org/wikipedia/commons/e/ed/DHA_numbers.svg

Typical 3H Radioactivity through the rat - % total dose Resveratrol in GI tract tissues, liver and blood

0

5

10

15

SI

Caecum

Colon

Liver

Blood

*

* *

3 hr- Tissues

% T

otal

Dos

e

0

5

10

15

SI

Caecum

Colon

Liver

Blood

*

* *

6 hr- Tissues

% T

otal

Dos

e

0

5

10

15

SI

Caecum

Colon

LiverBlo

od

12 hr- Tissues

% T

otal

Dos

e

0

5

10

15

SI

Caecum

Colon

Liver

Blood

* * * *

24 hr- Tissues

% T

otal

Dos

e

3H-Resveratrol: Mix 1:Tissues

Mix 1, Non Encaps- greenMix 1, Formulation 1- redMix 1, Formulation 2- blue

A B

C D

Augustin et al. JFF, 2011


- Probiotics



Process for encapsulation of probiotics

Oil

Protein

Carbohydrates

Emulsion

Probiotics

Mixing Reaction Drying

Film formed around bacteria

Extra coating (if necessary)

-Spray dry -Freeze dry

Excess bulk encapsulant


Encapsulation enhances survival during spray drying

1.0E-05

1.0E-04

1.0E-03

1.0E-02

1.0E-01

1.0E+00

1.0E+01

1.0E+02

Bifidobacterium

sp.

Lactobacillus sp.

Per

cent

sur

viva

l (Lo

g 10 s

cale

)Non-encapsulated

Encapsulated

102

101

100

10-1

10-2

10-3

10-4

10-5

Bif. In

fantis

Lacto

bacill

us

Acid

ofilu

s

**

**

** p < 0.01

Degree of protection during drying is strain dependent

Encapsulant Formulation: NaCas-Oligosaccharide-dried glucose syrup-oil (Heated-emulsion)


Enhanced survival at non-refrigerated storage

Encapsulation of probiotics (B infantis Bb-02) offers protection during storage

Crittenden et al. Appl Environ Microbiol.(2006)

10 μm

10 μm

Encapsulated probiotic

Non-Encapsulated

probiotic (●)

Caseinate-FOS-oil-DGS-B. infantis (▲);

Caseinate-FOS-oil-RS-B. infantis (○)


Probiotics survival during GI transition

Encapsulated B. infantis In SGF: pH 1.2, pepsin

p < 0.01

a

c

b

Bif. infantis

0.0001

0.001

0.01

0.1

1

10

100

1000

Non -

encapsulated

bacteria

Non -

encapsulated

bacteria plus

encapsulant

materials

Encapsulated

bacteria

10 2

10 1

10 0

10 - 1

10 - 2

10 - 3

10 - 4 Percent Survival (Log

10 scale)

0.0001

0.001

0.01

0.1

1

10

100

1000

Non -

encapsulated

bacteria

Non -

encapsulated

bacteria plus

encapsulant

materials

Encapsulated

bacteria

0.0001

0.001

0.01

0.1

1

10

100

1000

10 2

10 1

10 0

10 - 1

10 - 2

10 - 3

10 - 4

Perc

ent

Sur

viva

l

Viability of B. infantis after SGF

Non-

encapsulated

bacteria

Non-

encapsulated

bacteria plus

encapsulant

materials

Encapsulated

bacteria

Encapsulated B. infantis released in SIF: pH6.8, pancreatin

A

B

Encapsulated formulation: Caseinate-FOS-20%DGS-oil-B. infantis

Encapsulation enhanced the survival of probiotics during GI transition Crittenden et al. Appl Environ Microbiol. (2006)


Viability of spray dried microencapsulated LGG in apple juice

Augustin_Sanguansri CSIRO

LGG encapsulated in WPI; 4WPI:1RS;

1WPI:1RS; 1WPI:4RS; RS stored at

5°C

Integrity of microencapsulated LGG

formulations containing (A) WPI, (B)

1WPI:1RS, and (C) RS dispersed in

apple juice

• Viability: LGG in (WPI alone or in combination with RS) > RS

• WPI creates a buffered microenvironment within the hydrated colloid particle

• WPI protects embedded LGG from the low pH external environment

Ying et al. JFF, 2013

The Future of Microencapsulated

Bioactives

• Food industry • Demand for healthy bioactive ingredients continues to grow

• Incorporation of bioactives • Significant challenge remains for many bioactives isolated from their natural source

• Microencapsulation using milk proteins as encapsulants • Provides solutions for delivery of bioactives

Research and commercial industry partnerships

Benefits the food industry

Benefits the ingredient producer

Benefits the consumer


Thank you CSIRO Food & Nutrition Mary Ann Augustin Research Group Leader t +61 3 9731 3486 e [email protected]

emerging technologies & case studies - asaga · •the supercritical temp and pressure for co 2...

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