New Technology and Innovative

Probiotic Research into Human

Study in Indonesia

Endang S Rahayu
Faculty of Agricultural Technology
Universitas Gadjah Mada
endangsrahayu@ugm.ac.id

Endang S Rahayu
Universitas Gadjah Mada
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 Content of Presentation
• Introduction – Enterotype of Indonesian Gut
Microbiota
• Challenge for the development of indigenous
probiotics
• Probiotic foods – fermented products
• ‘Consortium-Research’ on Lactic Acid Bacteria
• Human Studies: market test, health benefits
• Conclusions
Endang S Rahayu
Universitas Gadjah Mada
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 Two different
Endang S Rahayu
Universitas Gadjah Mada enterotype based on
gut microbiota
1. Bifidobacterium/
bacteriodes (BB-
type) – China,
Japan, Taiwan
2. Prevotella (P-
type) – Indonesia
and Thailand
* Based on their
dietary habit
* Development
INDIGENOUS
PROBIOTIC is very
challenging to match
the gut microbiota
of each region
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 Challenge for the development of
indigenous probiotics
• Indonesia as one of the mega biodiversity countries, rich
in genetic resources
• Traditional fermented foods should have a great
opportunity to be carrier for probiotics
• All probiotic strains available in Indonesian market is
imported material
• Indonesian Society for Lactic Acid Bacteria and Gut
Microbiota (ISLAB-GM) has the capacity in developing
indigenous probiotics
• Consortium-Research (University, Research Institute,
Industry) should be established
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Universitas Gadjah Mada
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 Endang S Rahayu 5
Universitas Gadjah Mada
 Endang S.Rahayu
Universitas Gadjah Mada

Traditional fermented foods – Sources of food grade

microorganisms as starter cultures
 ● Fermented Foods (dadih, gatot, growol, etc) Preservation of
● Material intestine of healthy infant Cultures

Screening Characterization (Molecular)

●Resistance toward RAPD, 16Sr RNA, gen RecA, pepR
bile salt, gastric juice
●Antagonistic toward
pathogens
Thanks to:
Yakult Tokyo Japan, Dr. Koichi Watanabe
NBRC Tokoy Japan, Dr. Ken Ichiro Suzuki
Endang S Rahayu
Universitas Gadjah Mada
Products:
Cultures
●Lactobacillus paracasei SNP-2
●Lactobacillus platarum Dad -13;
●Mut-7; ●Mut-13, ●T-3
Strains were kept at:
FNCC – UGM, Indonesia
InaCC – LIPI, Indonesia
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NBRC – Tokyo, Japan
Phylogenetic tree based on 16S rDNA
Genus Lactobacillus of UGM
In collaboration with NBRC, Japan
(Miyashita Mika, Nanik Suhartatik, and E.S. Rahayu, 2012)

178
207
L. plantarum subsp. argentoratensis DKO 22T (AJ640078)
171
174A
L. plantarum subsp. plantarum NBRC15891T (AB326351)
177
Most Lactobacillus of Indonesian 173
182
fermented food and other 176
167A
166
sources belong to 170
172
Lactobacillus plantarum 190
183
179
181
Endang S Rahayu L. pentosus ATCC 8041T (D79211)
168
Universitas Gadjah Mada 983 175A
967 169
165B
1000 L. paraplantarum DSM 10667T (AJ306297)
900
L. fabifermentans LMG 24284T (AM905388)
L. xiangfangensis 3.1.1T (HM443954)
L. rhamnosus JCM 1136TT(D16552)
1000 L. zeae ATCC 15820 (D86516)
L. casei JCM 1134T (D16551)
0.01 995 L. paracasei subsp. paracasei JCM 8130T (D79212)
184
L. paracasei subsp. tolerans NBRC 15906T (AB181950)
1000 175B
167B
174B
165A T (AB626052)
L. fermentum NBRC15885
L. mucosae S32T T (AF126738)
1000 L. gastricus Kx156A7 (AY253658)
953 L. equigenerosi NRIC 0697T (AB288050)
L. ingluviei KR3T (AF333975) T
L. secaliphilus TMW 1.1309 (AM279150)
994 L. coleohominis DSM 14060T (AM113776)
Oenococcus oeni JCM 6125T (AB022924)
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 Functional Properties of LAB
• Proteolitic LAB to produce bioactive peptide (during food
fermentation) which important as ACE inhibitor (anti hypertension)
Wikandari and Pramono et al.
• LAB with high activity of α-galactosidase (could be used to ferment
soy-milk with aim to reduce un-digestable sugars , such as raffinose and
stachyose) – Titiek Djaafar et al.
• LAB with high activity of β-glucosidase to degrade isoflavone into a-
glycon form to increase the bio-avalability of this component as
antioxidant; this enzyme also play role in the degradation of
antocyanine into more active product – Nanik Suhartatik, et al.
• Folic acid producer of LAB (Siti Nur Purwandhani, et al)
• Uricase producer of LAB (Isti Handayani, et al)
• Bacteriosin producers LAB – for bio-preservative (Pediocin of
Pediococcus acidilactici F-11 for preservation of tofu) – Marwati et al
• Probiotic candidate (resistant to simulated gastric juice, bile salt and
have antagonisms toward pathogenic bacteria) and its application as
starter culture for production drink based on fermented milk.
Endang S Rahayu
Universitas Gadjah Mada
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Repetitive Extragenic Palindromic PCR with Primer BOX- A1R

14 days 28 days
Sources
Control Dad 13 Control Dad 13
Liver 1 (CL3) 1 (AL8) NI 2 (BL4, BL8)
Spleen 1 (CS3) NI 1 (DS2) 2 (BS3, BS4)
Kidney NI NI 1 (DK4) 2 (BK3, BK8)
Blood 1 (CB2) 1 (AB9) NI 3 (BB1, BB2, BB4)

BS3 BS4 CS3 DS2 AB9 BB1 BB2 BB4 CB2 Dad13 BK3 BK8 DK4 AL8 BL4 BL8 CL3

Translocation probiotic strain does not occur Endang S Rahayu

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Universitas Gadjah Mada
 Molecular detection of L.
plantarum Dad 13 (lane 6)
and strains isolated from
volunteer feces after
consumption (lane 8,9,11) -
identical
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Universitas Gadjah Mada
 Development of Probiotics
Type of Products
Food Bio-therapeutics
Traditional Functional foods Supplement Drug
fermented foods
Lactic fermented Novel-type Nutritional Selected viable cell
foods of dairy fermented foods supplements with for their-functional
products and and foods functional properties with the
plants, which still containing live cells properties clinical evidence for
contained live LAB with health Administered in therapeutics.
cells during properties powder (sachet or Administered in
consumed. Mostly dairy capsules) or tablet powder (sachet or
Cells are survive in products to maintain health capsules) or tablet
GI tracts and Fruits juices – condition
provide health probiotics
benefits to the host Cereal based foods

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Universitas Gadjah Mada
Probiotic (live cells) - Foods
Fermented Non Fermented Foods
Foods
Product type Wet Wet Dry – semi dry
Physiological Vegetative Vegetative Dormant
state
Products Yogurt Ice cream Dry milk powder Need High
Fermented Dairy desert Cereal based food Technology of
milk/drink Juices Confectionary Processing
Cheese Probiotic
Soya drink
Incorporation Cells increase Supplemented by Supplemented by
of cells during coated probiotics coated probiotics
fermentation
Shelf-life Refrigerated Refrigerated Ambient Temp.
Up to 2 months Up to 2 month 12 months
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Universitas Gadjah Mada
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 Probiotic
Probiotic
Starter culture
Products
106 CFU/g (ml)
Fermentation Fermentation
i
108 CFU/g (ml)
Boimass

PprotectionI / Coating
(micro encapsulation)

Drying
(freeze / spray)

Probiotic Powder 1010-12 CFU/g (ml)

Mix with food

Fermented Product Functional food Suplements

Probiotic Probiotic
107-8 CFU/g (ml) 107-8 CFU/g
107-8 CFU/g

Endang S.Rahayu
Fak. Teknologi Pertanian UGM
 Tape
Peanut Brem Ketan
Lactic Drink Tape Probiotic
Probiotic
Ketan POWDER
Probiotic Lactose
Reduction of AFB1 intoleranse
(Binding LAB-AFB1)
Increase LAB in GIT
Lower Enterobacteriaceae Yogurt
Soy Lactic Starter Cultures
Drink
Local INDUSTRIAL
Probiotics TRIAL
SNP-2 Fermented
Dad-13 DRINK
Mut-7; Mut-13
Antagonistic T-3
toward
pathogens Encapsulation
Spray Drying Ca-Alginat
Maltodextrin
Extrusion
Probiotic Research Emulsion
at Univ. Gadjah Mada Centerfill Juices
Rahayu ES, et al., 2009 Manggo Ice Cream
Candy Pine apple Viability stable
70C, 10 min 8 weeks
Decline 2Endang
Log Cycle
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Universitas Gadjah Mada
 Challenge in starter culture production
(Tyas Utami, et al., in progress)
• In Indonesia ‘halal’ food is very important
• Food Industries need their raw material
(including starter culture) that have halal
certificate
• Challenge – development of halal starter
culture using media containing organic
material

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Basic and Applied Pre-
Research commercialization Commercialization

University University and Industry Industry

Commercialization of Research Products

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Universitas Gadjah Mada
Research Consortium Lactic Acid Bacteria

Name Institution Field

1 Prof.Dr. Endang S. Faculty of Agricultural Technology PIC, Lactic acid bacteria
Rahayu Universitas Gadjah Mada
2 Dr. Tyas Utami Faculty of Agricultural Technology Starter cultures
Universitas Gadjah Mada
3 Dr. M. Nur Cahyanto Faculty of Agricultural Technology Fermentation
Universitas Gadjah Mada
4 Prof.Dr. M Juffrie Faculty ofMedicine Human trial
Universitas Gadjah Mada
5 I Nengah Sujaya PhD Universitas Udayana Health benefit
6 Dr. Achmad Dinoto Lembaga Ilmu Pengetahuan Culture Collection
Indonesi, LIPI - INACC
7 Ir. Sri Nurfiani PT Yummy Food Utama, Jakarta Application of LAB to
industry (fermentation)
8 Ir. Eni Zulaichah PTEndang
YummySFood
Rahayu
Utama, Jakarta 18
Universitas Gadjah Mada
Production of Powder
of Starter Cultures
Endang S Rahayu
Universitas Gadjah Mada
Laboratory
Strain of LAB
indigenous Drink based on
Fermented Milk
Factory

Cell Formulation of Drink

Propagation

Centrifugation

Freeze Drying Powder of

Starter Culture Fermentation of Milk
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Road-Show PROBIOTIC Day – MARKET test, Yogyakarta, Denpasar, Surabaya

Aroma
4
suka/tidak 3 Mouthfeel
suka 2
1 131
0
Lingering 379
Kemanisan
aftertaste

Kekompaka
Keasaman
n

Isolat UGM PT Yummy

Aroma
4.00
Suka/Tidak 3.00
Mouthfeel
suka 2.00
1.00
0.00
After-taste Kemanisan

Endang S Rahayu Keasaman Kekompakan

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Universitas Gadjah Mada
 Advantage and Limitation
(Industrial-side report)
Advantages
• Consumer acceptance is good
• A unique taste and delicious and in accordance with
consumer tastes Indonesia.
• Viscosity better
• High viability of probiotic cells in the product

Limitation
• Longer fermentation time
• Inconsistent during fermentation
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 Human Trials
• Need a large number of sample studied
• Must be industrial product to assure their
safety
• Collaboration with industry is needed – to
produce the samples studied
• Health benefits for indigenous probiotic (on
going research): anti-influenza, anti-allergy,
lipid profile, blood pressure, and uric acid
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Indonesian Society for Lactic Acid Bacteria and
Gut Microbiota (ISLAB-GM), since 2003

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 Conclusions
• Indigenous bacteria have the potential to be
developed as a starter culture for fermentation-
based industries
• Support from industry (in term of the production of
sample studied) is necessary to carry out the human
study
• Research consortium consist of university, research
institute and industry is needed for pre-
commercialization and commercialization stages

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THANK YOU

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Universitas Gadjah Mada