An overview of my research in a Centre of Research Excellence in New Zealand to develop novel technology which successfuly enabled probiotic strains remain stable for longer duration under ambient storage conditions.

1. Shelf stable probiotic foods –
Now a reality Arup Nag
29.06.201129.06.2011

2. Arup Nag
29.06.2011
Arup Nag
29.06.2011
Agenda
Identifying the opportunity
Core technology
Comparison with selected established technologies
Path to commercialization

3. Health benefits of probiotics are:
•Immunity enhancement
•Improved gut health
Prevention of intestinal disturbances
Balancing intestinal micro flora
Prevention of various types of diarrhoea
•Prevention of certain types of allergenic reaction in infants
•Improved oral health
Enormous body of scientific research to show the health benefits of probiotic bacteria
Most common genus: Lactobacillus and Bifidobacterium
What are probiotics?
Probiotics are live microorganisms which when administered in adequate amounts
confer a health benefit on the host (FAO/WHO, 2001)

4. Probiotics market
Probiotics market is approaching close to 20 billion dollars.....further growth possible with
technology that helps reach new consumers

5. 20072000
Yogurt
Only
Yogurt, fermented milk, cottage cheese, soy
and more
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Sales(BillionEuro)
2001
2002
2003
2004
2005
2006
2007
Source:‘Danonedairy–theaccelerationofgrowth’presentationbyBernard
HoursinDanoneinvestorseminar2007
Source:MintelGNPD
Danone Activia is one of the most successful food product in recent time – this is positioned as
digestive food based on scientifically proven probiotic bacteria - Activia grew over 600% in 7 years
Success of Activia demonstrates the high potential of probiotic as a sustainable and highly credible
functional ingredient
Review of secondary market data

6. Hard to compete with the established players within the existing categories ........... huge
opportunity in extending offerings from chilled shelf to ambient shelf
Technology for delivering
probiotics in shelf stable foods
Technology gap
Opportunity in probiotics area
Enabling technology with sufficient technical insulation to unlock untapped
opportunities

7. Consumption of probiotic bacteria 108 – 109 cfu/g per day for health benefits
Considering consumption of 100 g probiotic rich food per day, probiotic rich food
needs to contain 106 – 107 cfu/g viable cells of probiotic bacteria
Challenges in maintaining viability of probiotic cells during processing, storage
and gastro-intestinal transit
Application in probiotics restricted to chilled yoghurt, fermented milk and juice
Limited technology available for shelf stable food and beverage
Challenges in delivering probiotics
Processing
•High temperature
•Low water activity, High acid
Storage
•Time, Temperature
•Low water activity , High acid
•High acidity in stomach
•Bile salt in intestine
GI tract
Harsh conditions during processing, storage and GI tract transition pose challenges in
delivering probiotics for functional benefits

8. Agenda
Identifying the opportunity
Core technology
Comparison with selected established technologies
Understanding the intricacies
Path to commercialization
8

9. ProbiolifeTM is a novel technology to improve the stability of probiotic bacteria
over a long period of storage at 25⁰C
Probiotic strain
Grow
Encapsulate
Drying
Most of the common probiotic strains (e.g. Lactobacillus &
Bifidobacterium) can be used
Growth is under specially designed condition
Encapsulate in an optimised matrix – consisting of only
natural ingredients
Drying using a special type of equipment with optimized
conditions
ProbiolifeTM Technology - an insight

10. 4
5
6
7
8
9
10
11
0 4 8 12 16 20 24 28 32 36 40 44 48
LogCFU/GmDrySolids
Storage at 25ºC (Weeks)
L. Casei CRL431 cells stored at ambient temperature maintained viability at over 1 billion live
cells per gram after 9 months and over100 million live cells after 1 year.
Storage Stability
Improved storage stability of probiotic bacteria at ambient temperature
1 Billion

11. Understanding the Architecture
Bacteria L.casei CRL 431
Encapsulation in matrix
Matrix surface closer look
SEM images to elucidate the morphology of the matrix

12. ProbiolifeTM technology developed with L. casei CRL431, and then tested with a number of
Lactobacillus and Bifidobacterium strains.
For all the 8 strains tested ProbiolifeTM technology gave far better stability to the cells compared to
freeze drying during long term storage at ambient temperature.
Lactobacillus acidophillus ATCC 4356 Lactobacillus rhamnosus ATCC 53103
Bifidobacterium lactis BB12 Lactobacillus casei ATCC 393
Validation of ProbiolifeTM using different probiotic strains
ProbiolifeTM technology provided enhanced stability to the cells compared to freeze drying
during long term storage at ambient temperature.
Bifidobacterium lactis BB12 Lactobacillus casei ATCC 393

13. ProbiolifeTM technology provided enhanced protection to the cells during simulated gastro-
intestinal transit
Simulated gastric fluid at pH 2.0 with 0.32%
pepsin.
Free cells reduced to undetectable level after
90 min
Encapsulated cells reduced by 4.5 log cycle
Incubation with 1% bile extract at pH 6.8.
Free cells reduced by 2.7 log cycle.
Encapsulated cells reduced by only 0.6 log
cycle
Stability in gastric & intestinal fluid

14. The physical properties of the ProbiolifeTM ingredient is comparable with common dairy
ingredients making it feasible for wider range of applications
ProbiolifeTM
Whole Milk Powder
Skim Milk Powder
ProbiolifeTM physical properties
D32 : 125.6 µm
Aw : 0.27
Bulk Density: 470 Kg/m3
ProbiolifeTM
Whole Milk Powder
Skim Milk Powder
Physical characterization

15. ProBioLife TM can be applied to a range of food products
ProBioLifeTM powder
Shelf stable foods Shelf stable powder
beverage
Conform WHO/FAO recommendation 1 – 10 million live cells/gram....100 million cells per serving
ProBioLifeTM – application in consumer food products
RTD beverages
Application using
dosing cap or like

16. 4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
0 4 8 12 16 20 24
LogCFU/Gm
Storage at 25ºC (Weeks)
ProbiolifeTM fortified shelf stable food products maintained cell viability during storage
Shelf stability of ProbiolifeTM fortified products
L. casei CRL431 cells in encapsulated form were added to cereal bar, malted beverage and chocolate spread.
The products were stored at 25ºC for 6 months
The loss in viable cell populations were recorded as 1.0, 0.2, 0.8 log CFU/gm for Powdered
drink, Chocolate spread and Cereal bars respectively.

17. Added
Original
cell After After Final mix
water population 5 Min 30 Min
temperature
(⁰C)
temperature
(Log
CFU/gm)
(Log
CFU/gm)
(Log
CFU/gm) after 30 min
At 75⁰C 8.4 7.9 7.3 35
At 65⁰C 8.4 8.3 7.9 25
At 55⁰C 8.4 8.5 8.4 25
Some consumers like to mix powder dairy beverage with hot water .....test confirmed high
level of viable cells of L. casei after mixing with hot water
ProBioLifeTM – application in powdered dairy beverage

18. 5.4
5.6
5.8
6
6.2
6.4
6.6
6.8
7
Color Flavor Taste Overall
Acceptabilityscore
Triangle test could not identify any difference between 2 chocolate flavour powder
beverage fortified with or without Probiolife ingredient
No negative impact of Probiolife ingredient fortification on the acceptability of powder
beverage
ProBioLife TM - application in powdered dairy beverage
Reconstituted chocolate flavoured powdered beverage
Reconstituted chocolate flavoured powdered beverage with ProBioLife ingredient
ProBioLifeTM ingredient does not impact the flavour of the food product

19. Agenda
Identifying the opportunity
Core technology
Comparison with selected established technologies
Path to commercialization
19

20. Comparison of ProbiolifeTM technology Vs other established technology
4
5
6
7
8
9
10
11
0 4 8 12
LogCFU/Gm
Storage at 25oC (weeks)
Novel Technology
Commercial freeze dried
Freeze drying in same
matrix as NT
Spray drying in same
matrix as NT
Commercial freeze dried granules were most affected during ambient storage
Freeze drying and spray drying process using ProbiolifeTM matrix showed slight improvement
Stabilization using ProbiolifeTM technology offered significantly better stability
ProbiolifeTM technology provided enhanced stability to bacterial cells compared to other
popular stabilization technologies (freeze drying and spray drying )

21. Agenda
Identifying the opportunity
Core technology
Comparison with selected established technologies
Path to commercialization

22. ProBioLifeTM – cost comparable to many standard functional ingredients
ProBioLifeTM powder competitive advantages
Probiotic strain
Grow
Encapsulate
Drying
Assumed: $500/kg as
freeze dried culture
$10/kg ProBioLife powder
(Assumption: $2.5/kg for
processing and packaging)
*Licence fee and capital investment not included
Competitive advantage:
Not strain specific
Natural/GRAS
Cost effective
3 – 4 cents per serving for a
powdered dairy based beverage

23. First provisional patent
(Australia) – August 2010
Second provisional patent
(New Zealand) – April 2011
PCT – August 2011
Protecting the IP

24. ProBioLifeTM Consumer products companies
ProBioLifeTM B2B ingredient companies
Model 1
Model 2
ProBioLifeTM Co-packerModel 3
Consumer products companies
Culture supply
Consumer products
companies/ingredient companies
Model 1:
Abbott - Asia Pacific
GSK - India
PepsiCo – USA/ AMEA
Kraft – USA
Sanitarium - Australia
Model 2:
Danisco - Finland
Chr. Hansen – Denmark
DSM – Asia Pacific
BLIS – New Zealand(ABPL)
Need to work out value chain for every model
Commercialization models
Three potential business models

25.  Prof. Harjinder Singh-Co-Director, Riddet Institute , Massey University, Palmerston North, New Zealand
 Dr. Shantanu Das- Product Development Manager, Riddet Institute , Massey University, Palmerston North, New Zealand
 Mr. Arup Nag- Food Technologist, Riddet Institute , Massey University, Palmerston North, New Zealand
 Mr. Devastotra Poddar- PhD Student, Riddet Institute , Massey University, Palmerston North, New Zealand
 Mr. Sumon Saha- PGDip. Student, SEAT, Massey University, Palmerston North, New Zealand
 Mr. TK Vyas- PGDip. Student, IFNHH, Massey University, Palmerston North, New Zealand
 Mr. Jacob Lollike- Bachelor of Food Science ,University of Copenhagen ,Denmark
 Ms. Beibei Zhou- MSc ( Food Technology), Wageningen UR (University and research centre), Netherlands
 Ms. Esther Ang- Bachelor of Food Technology, Massey University, Singapore campus, Singapore
 Ms. Hui Kheng-Singapore Polytechnic, Singapore
 Ms. Marrie Camilie- Intern from France
 Prof. John Bronlund - Professor, SEAT, Massey University, Palmerston North, New Zealand
 Dr. Jon Palmer- Lecturer ,IFNHH, Massey University, Palmerston North, New Zealand
Contributors to the work
Acknowledgements
Prof. Paul Moughan for encouragement, motivation and valuable advice
Massey University Research Services for support
Mr. Rick Gain and Mr. Richard Cartis for valuable advice
Mr. Mark Ward for Encouragement and support
AJ PARK for help in Patenting
Manawatu Microscopy Services, IFNHH & SEAT for technical support
All Riddet Institute staff members for help and support