Probiotics as Functional Foods

Ingrid S Surono
Food Technology Department
BINA NUSANTARA UNIVERSITY/Indonesian Scientific Society
for Probiotics and Prebiotics
Second ASIA Food and Beverage Summit 2016, Nov 7-8

Presentation Outline :
Introductory Remarks
Current Situation
Key Drivers
Current Situation : probiotic Functional Food
products
Government Policies
Concluding Remarks
Second ASIA Food and Beverage Summit 2016, Nov7-8

Introductory Remarks

Second ASIA Food and Beverage Summit 2016, Nov 7-9

(Glausiusz J. Discover June 2007)

Factors affecting probiotic survival

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Current Situation : probiotic

Functional Food products

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Functional Foods and Nondairy Probiotic Food Development: Trends, Concepts, and Products

Comprehensive Reviews in Food Science and Food Safety

Volume 9, Issue 3, pages 292-302, 29 APR 2010 DOI: 10.1111/j.1541-4337.2010.00110.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1541-4337.2010.00110.x/full#f1

Foods: dairy and many others

Probiotics

Storage

Inoculation

FF techniques

15

Probiotic Chocolate

Second ASIA Food and Beverage Summit 2016, Nov 7-8

potential as probiotic carriers : Mayonnaise,

soymilk, meats, baby foods, ice creams, fruit
drinks, vegetable drinks, coconut water,
cereals

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Key Driving Forces For Probiotic

Development in Indonesia

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Probiotic : Functional foods

Challenges
Growth of elderly population
Increase of lifes length
More health conscious
Increase interest in prevention than
treatment
Consumer awareness

Figure 10. Six Dynamic Markets : China, India, Indonesia,

Brazil, Kenya and Pakistan

Source : Tetra Pak, 2012

Figure 12. Low-Income Consumers are the

next big opportunity for the dairy industry
They represent 38% of LDP in developing countries

ToP = Top of the Pyramid-above $8/day

DiP= Deep in the pyramid-$2 to $8/day
BoP= Bottom of the pyramid-below $2/day

Source : Tetra Pak, 2012

Milk Consumption Growth in

Indonesia

Based on gender :

POPULATION IN
INDONESIA OPPORTUNITY

50,47%

Source : BPS, 2010

49,63%

Male
Female

Demographic bonus for

Indonesia

Working group 170,9 mo in 2015,

reach 195,2 mo in 2040 decline to
191,5 in 2050

Children younger than 15 years

decline but still at 50 mo in 2050,

Eldery increase slowly up to 2035,

Trend in number of children, working-age and older

persons, Indonesia, 1950-2050
250

Population in millions

200
working-age
150

Increase remarkably tp 49,6 mo in

2050, equal to children population

100
children 0-14
50

50
20

40
20

30
20

10

00

20
20

20

20

90
19

80
19

70
19

50

60
19

19

Windows opportunity : 2020-2030

older persons 65+

Year

30/09/2011

sm adioetomo/S2KK_Pop_DEV

26

Government Policies For Dairy

Development in Indonesia

Second ASIA Food and Beverage Summit 2016, Nov 7-8

FAO/WHO Guidelines as a probiotic, published 2002

Food and Agriculture

Organization of the United
Nations

Strain identification by phenotypic and

genotypic methods
Genus, species, strain
Deposit strain in international culture collection

Strain(s) in specific product formulation

Safety assessment
In vitro and/or animal
Phase 1 human study
Double blind, randomized, placebo-controlled
(DBPC) phase 2 human trial with sample size
and primary outcome appropriate to determine
if strain/product is efficacious

Preferably second
independent DBPC
study to confirm
results

True
Probiotic

World Health Organization

Providing a definition

Strain selection and characterization

Strain dependent effect : subject, Vulnerable ?
Mechanisms
Genetic stability
Adverse events/morbidity, mortality

probiotic should properly identified, documented, manufactured under good

manufacturing practices or proven clinically

Claims should mention :

Genus, species and strain (physiological effect depend on
specific strain),
Minimum viable numbers of the probiotic at the end of the
shelf life,
Suggested serving size to be consumed in relation with the
health claim,
Storages conditions,
Reachable customer service to get information

probiotics in food products :

Probiotics should be viable in the consumer product
Standard for health claims: 106 107 cfu/g (FAO, WHO, 2001)
Industry is interested in using existing strains in new
applications
Regulations for functional foods get more strict, and
health benefits need to be demonstrated in human
studies.
Many probiotic products presently on the market do
not meet these criteria

Potential Indigenous
Novel Probiotics

Second ASIA Food and Beverage Summit 2016, Nov 7-8

TRADITIONAL WAY OF DADIH MAKING

Fresh raw unheated buffalo milk, poured into bamboo tubes

DADIH
Example of Indonesian traditional fermented raw
buffalo milk of West Sumatra

The followings are selected findings on probiotics

isolated from Dadih

Molecular Identification
Lactobacillus plantarum IS-10506
TCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGG
GAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATG
CTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAA
GATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGC
TAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCG
ACCTGAGAGGGTAATCGGCCACATTGGGGACTGANACACGGCC
CAAACTCCTACGGGGAGGCAGCAGTAGGGAATCTTCCCCAATG
GGACGAAAGTCTGATGGA
This sequence shows 98% homology with different L. plantarum sequences
held in genbank (DQ239698.1; DQ239695.1; AY675256.1; AL935261.1
and others), so it can be identified as L. plantarum.
16S rRNA gene sequence as Lactobacillus plantarum IS-10506 (GeneBank
accession no DQ860148

4th International Symposium on Probiotics and Prebiotics 2016

Journal of Food Science, Vol. 72, No. 3,

2007, M89-M93
MC Collado, IS Surono, J Meriluoto and
S Salminen

% adhesion

The improvement of Intestinal vili induced by probiotic

LIS 10506 and LIS 20506
A:
B:
C:
D:

Normal Vili
Disruption of vili caused by LPS E.coli
the improvement vili by LIS 10506
the improvement vili by LIS 20506

Reza Ranuh, 2008

Ekspresistructural
Protein
Border
Usus
Intestinal
brush Brush
border
protein
Expression Distribusi
of intestinal
brush
border protein

Tingkat Ekspresi

30
25
p<0,05)

20

KONTROL

p<0,05)
p<0,05)

LPS control
LPS + LIS 10506

p<0,05)

15
10

P>0,05)

LPS + LIS 20506

0
D

J
G

J
M

ZO

Protein Brush Border Usus

O
Galectin-4, Myosin-1a,
ZO-1 and Occludin

Reza Ranuh, 2008. PhD dissertation, Medical Faculty,

Airlangga University

TH1, Treg and TH2 immunoregulation through TLR2 and TLR4 by

Lactobacillus plantarum IS-10506 (LIS) probiotics in reducing
allergic reaction. Experimental Study in Mice Subjects

LIS probiotics group

Control group

TH1 > Treg > TH2

TH1<Treg <TH2
Allergic
Reaction

Adaptive
Immunity

Innate
Immunity

Study on animal model (Balb/c mice)

(Endaryanto A, Subiyanto and

Surono, IS, 2006)

Delta fecal sIgA

(mg/ml)

Effect of probiotic strain on fecal sIgA

0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000

0.647

0.491
0.417

0.407

0.319

0.311
0.203 0.196
0.102
0.021

0.026

0-3 days

0-7days

0.052
0-10 days

Treatment periods
A0

A1

A2

A3

(p=0.05), *significantly different

Ao : control, A1 : L. casei Shirota, A2 : L. plantarum IS-10506,
A3 : L. plantarum IS-20506

Animal experiment Balb/c mice

Endaryanto, A, Subijanto, MS,

Surono, IS, 2006
p=0.000
p=0.000

We need more full circle research

probiotic E. faecium IS-27526 has significant positive

effects on humoral immune response, salivary sIgA, in
underweight preschool children, and on weight gain of
pre-school children

A challenging period is
opened
New prospective routes Validated scientific
for probiotics
methods can be (and
should be) transferred
Increased need for good
science and
from one area to
another
good quality studies
To answer regulatory
Supporting outcome
requirements
from different
To reassure consumers
laboratory
To boost business
Requirements on
opportunities on solid
scientific evidence for
and long-term bases
marketing

Shelf stability
High stability at room
temperature: possible
but very difficult to
obtain

http://www.institut-rosell.com/

Several reports of
false CFU counts in
commercial products
Probiotics

Storage

Inoculation

FF techniques

55

Effect of format on culture delivery

Lactobacillus GG found in GI tract
1011
Total in the portion
In feces, per g

Population (UFC)

1010
109
108
107
106
105
104
103
1

Powder

Probiotics

Fermented milk Milk

(200 mL) (200 mL)

Storage

Cheese
(30 g)

Inoculation

Fruit juice
(200 mL)

FF techniques

Saxelin et al., (2003) Dans:

Functional dairy products,
CRC Press - Woodhead Publ.
Ltd., Mattila-Sandholm &
Saarela Eds., p7

56

Storage: effect of encapsulation

Non-fat dry milk
Stability (%) P. acidilactici

Time
(months) Standard
FD

Encapsulated by
spray-coating

100

100

42

85

38

73

32

71

15

69

Suita-Cruce & Goulet 2001. Food Technol. 55(10): 36-42

Probiotics

Storage

Inoculation

FF techniques

57

Stability during storage

Effect of
temperature

10 X

Effect of
strain

10 X

Champagne et al. (1996) Food Res. Internat 29: 555-562.

Probiotics

Storage

Inoculation

FF techniques

58

Effect of freeze-drying matrix

Stability of L. rhamnosus E800 during freeze-drying (FD) and storage of powders at an

elevated temperature (37C). Different carriers were used as cryoprotectants in FD.

Saarela et al (2006) Internat J. Food Microbiol. 112: 171-178.

Probiotics

Storage

Inoculation

FF techniques

59

 Dried products are not stable at room temperature:

need for technology and storage practices
Rehydration conditions are critical in using dried
products. This must be considered when dried
cultures are:
added to foods and beverages
analysed for CFU counts
used in clinical trials

Probiotics

Storage

Inoculation

FF techniques

60

CFU counts
Clinical:
best to use new product
for each assay. If not
possible
dont assume opened
sachet/bottle has constant population
and/or strain ratio during storage.
Carry out tests. Easier if you have
single strain cultures.
Second ASIA Food and Beverage Summit 2016, Nov 7-8

61

Protective ingredients
Not always the best
protective compounds
can be used
Great constraints with respect to:
Religions

(Ex. Kosher)

Allergies (Ex. Dairy, Gluten, Soy )

Partially explains variable stabilities
Second ASIA Food and Beverage Summit 2016, Nov 7-8

62

Challenges in the development of a

functional food
# 1 : Selection of the ingredient
# 2 : Concentration of active ingredient to add
# 3 : Toxicity
# 4 : Method of incorporation into the food
# 5 : Analysis of active components
# 6 : Stability during storage
# 7 : Effect on sensory properties
# 8 : Health claim
Second ASIA Food and Beverage Summit 2016, Nov 7-8

63

Direct to the vat inoculation (DVI)

Bulk starters may still be produced at the plant
but generally not bulk probiotics

Why ?
Requirement for a given number of cells
Need

for standardization - easier with DVI

Generally do not grow well in milk and with starters

Might need own production media and tank

Capricious organisms (oxygen )

Second ASIA Food and Beverage Summit 2016, Nov 7-8

64

Concluding Remarks

Second ASIA Food and Beverage Summit 2016, Nov 7-8

Key messages
The gut microflora is generally important for maintaining
good health
Probiotic may be used to improve health and reduce
undesirable activity of the gut microbiota
Indigenous probiotic has been exposure to microbes
surrounding, more suitable for local people
Recommendation of 109 - 1010 CFU as effective dose for a
probiotic , strain dependent

Negative or bad
results?
Regulatory people and Scientists:
need to be careful before stating
counts in commercial products
Probiotics require specialised CFU methodolology
Classical Plate Count Agar may underestimate counts
Rehydration condition affect results
Free encapsulated products
Variable strain results on selective/differential media
Need for strain ID (molecular tools)

Suggest you
contact the
manufacturer

67

 Lactobacillus plantarum IS-10506 and Enterococcus faecium IS27526 of dadih origin are potential novel probiotic which
showed effect in both in vivo and pilot human study.
Supplementation of Enterococcus faecium IS-27526
to
children younger than 5 years significantly increased
bodyweight of apparently healthy children and significantly
increased serum IgA of undernourished children
The supplementation of microencapsulated Lactobacillus
plantarum IS-10506 at 1010 cfu/day significantly increased
fecal sIgA in probiotic group (p<0.01) and in combination of
probiotic and zinc group (p<0.027) after 90 days to children
aged 12-24 months. No adverse event was observed in both in
vivo and in human.

100 years ago, Metchnikoff said we should eat lactic acid

bacteria for long life. What is Long Life?
Average life span is:
<60 years for men in Russia and falling
57 years for Australian Aboriginal men
<60 years for New Zealand Maori
33.9 years Botswana
34.5 years Lesotho
81.15 years for Japan
Diabetes:

171 million in 2000 expected to be 366 million in 2030

Japan one of lowest at <5%

Overweight/Obesity:
39.8 million (more than 57%) American adults are overweight (CDC)
We need to share our research with ALL the world

YES

which bacteria do we eat today?

Let food be your medicine and
medicine be your food

Your kind attention is highly appreciated