Czech University of Life Sciences Prague Faculty of Agrobiology, Food and Natural Resources

Current Research and Use of Probiotic, Prebiotics and Synbiotics

Prof. Ing. Vojtch Rada, CSc. Department of Microbiology, Nutrition and Dietetics
prof. Ing. Vojtch Rada, CSc. rada@af.czu.cz

PROBIOTICS PREBIOTICS SYNBIOTICS

 PROBIOTICS
Life microorganisms which when administered in adequate amounts confer a health benefits on the host (FAO/WHO, 2003)

PREBIOTICS

A nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon and thus improves host health (Gibson and Roberfroid, 1995)

SYNBIOTICS
Combination of probiotics and prebiotics

Changes in faecal flora during life (Mitsuoka, 1992)

Protective role of probiotics (Fuller, 1989)

Newborn

Limited acces to mother

Full acces to mother

Incomplete protection

Deficient microflora

+ Probiotics

Complete microflora Protection

Probiotic bacteria
Lactic acid bacteria
Lactobacillus acidophilus Lactobacillus casei Lactobacillus rhamnosus Lactobacillus salivarius Lactobacillus plantarum Lactobacillus delbrueckii ssp. bulgaricus Lactococcus lactis Enterococcus faecium Streptococcus thermophilus Pediococcus pentosaceus

Bifidobacteria
Bifidobacterium animalis ssp. lactis Bifidobacterium longum Bifidobacterium bifidum Bifidobacterium breve Bifidobacterium infantis Bifidobacterium pseudolongum Bifidobacterium thermophilum

Other bacteria
Escherichia coli Bacillus sp. Clostridium butyricum

Fungi
Saccharomyces sp. Aspergillus oryzae Candida pintolopesii

Commercially available probitic organisms

Lactobacillus acidophillus LA5 (Chr. Hansen) Lactobacillus rhamnosus GG (LGG; ATCC 53103; Gefilus) Lactobacillus casei Shirota (Yakult) Lactobacillus casei Imunitass (Danone)

Commercially available probitic organisms

Bifidobacterium animalis subsp. lactis DN173010 (Danone) Bifidobacterium animalis subsp. lactis BB 12 (Chr. Hansen) B. longum BB536 (Morinaga Milk Industry, Japan) B. breve Yakult (Yakult, Japan)

Mechanisms of Probiotics
Adhesion to intestinal mucus and epithelium Combined probiotics and patogen aggregation Production of antimicrobial substances Immune effects of probiotic bacteria Modulation of gut microbiota and their metabolites

Autoaggregation
Bifidobacterium spp.

Clostridium spp.

Coaggregation
Bifidobacterium spp. + Clostridium spp.

Prebiotics
FOS fructooligosaccharides GOS galactooligosaccharides SOS soya oligosaccharides XOS xylooligosaccharides MOS isomaltooligosaccharides HMO human milk oligosaccharides

Criteria for prebiotics:

Resistance to gastric acid and intestinal enzymes, no absorption in the gut Fermentation by intestinal bacteria Selective stimulation of the growth and /or activity of specific bacteria Target bacteria: bifidobacteria, lactobacilli

FOS - Fructooligosaccharides

GFn type

Fn type

Inulin (FOS) content in selected plants (g/100 g) (Ebringer, 2002)

Plant inulin content Onion Garlic Leek Chicory Sweet potatoes (Tuberus) Dandelion Banana in g/100g 27 9 16 3 10 30 47 16 20 12 15 0,3 0,7

FOS (oligofructose) Fructooligosaccharides in infant formulas

GOS - Galactooligosaccharides

GOS in infant formulas

GOS/FOS = 9:1

SOS Soya oligosaccharides

Raffinose series oligosaccharides (RSO)

raffinose (n = 1) stachyose (n = 2) verbascose (n = 3)

Stachyose and raffinose content in leguminoses (in % of dry matter; Velek, 1999)
Plant Bean Pea Lentil Soya Raffinose 0.3 1.1 0.6 1.0 0.3 0.5 0.2 1.8 Stachyose 3.5 5.6 1.9 2.7 1.9 - 3.1 0.02 4.8

Monomers of human milk oligosacharides

D-glucose (Glc) D-galactose (Gal)

N-acetylglucosamine (GlcNac)

L-fucose (Fuc)

Sialic acid (N-acetyl neuraminic acid, Neu 5Ac)

Basic structure of HMO

Fucose

Vazba
etzec

Vazba

Neu5Ac

Tabulka Jednotliv druhy oligosacharid jejich struktura a koncentrace v mateskm mlce (Warren et al., 2001) Zkratka Triviln nzev Struktura Lac Laktza Gal(14) Glc 2-FL 2-Fukosyllaktza Fuc(12) Gal(14) Glc 3-FL 3-Fukosyllaktza Gal(14) Glc Fuc(13) LDFT Laktodifukotetraza Glc LNT LN/T LNF-I LNF-II Fuc(13) Lakto-N-tetraza Lakto-N-neotetraza Lakto-N-fukopentaza Lakto-N-fukopentaza II Gal(13) GlcNAc (13) Gal(14) Glc Gal(14) GlcNAc (13) Gal(14) Glc Fuc(12) Gal(13) GlcNAc (13) Gal(14) Glc Gal(13) Fuc(14) LNF-III Lakto-N-fukopentaza III GlcNAc (13) Gal(14) Glc Fuc(13) LDFH-I Lakto-N-difukohexaza I GlcNAc (13) Gal(14) Glc LDFH-II Fuc(14) Lakto-N-difukohexaza II GlcNAc (13) Gal(14) Glc Gal(13) Fuc(14) Fuc(12) Gal(13) Gal(14) Fuc(12) Gal(14)

GlcNAc (13) Gal(14) Glc

MFLNH-III

Fuc(13) Monofucosyllakto-N-hexaza III

Fuc(13)
Gal(14) Glc Fuc(13) GlcNAc (16) Gal(14) Glc Fuc(12) Gal(13)

GlcNAc (16) Gal(14) Gal(13)GlcNAc (13) DFLNHa Difukosyllakto-N-hexaza a Gal(14) GlcNAc (13)

Pongo abelii Gorilla gorilla

Pan troglodytes Pan pansicuss

Functions of HMO
Prebiotic function bifidobacteria Development of CNS (sialic acids) Against pathogens (prevence of adhesion) Resorption of minerals (Ca, P) Other?

Synbiotics = combination of probiotics and a prebiotics

Current topics in probiotic and prebiotic research

Are probiotic and prebiotic save? Is mother milk a source of living probiotic bacteria? Effective dosage for probiotic effect? Probiotics and legislation

Are probiotics and prebiotics save?

Probiotics shloud not be administered to preterm infants and individuals with serious diseases Lactobacilli produce D- lactate could be even toxic for chicken (people) Prebiotic could cause diarhoea in infants Prebiotics could support nonprobiotic bacteria

Probiotic bacteria in human milk

Martn R., Langa S., Reviriego C., Jimnez E., Marn M.L., Xaus J., Fernndez L., Rodrguez J.M.: Human milk is a source of lactic acid bacteria for the infant gut. J. Pediatr. 143, 754-758, 2003 Sinkiewicz G., Nordstrom E.A.: Occurence of Lactobacillus reuteri, lactobacilli and bifidobacteria in human breast milk. Pediatr. Res. 58, 415, 2005 Gueimonde M., Laitinen K., Salminen S., Isolauri E.: Breast milk: a source of bifidobacteria for infant gut development and maturation? Neonatology 92, 64-66, 2007

Bifidobakteria in Human Milk

Human Milk without Bifidobacteria

Detection of Bifidobacteria in Human Milk using Fructose-6-Phosphate Phosphoketolase Tests

Effective dosage for probiotic effect?

Daily dose (1O7- 1010 CFU) Daily frequency of administration (1-4 times) Timing of administration Duration of administration (1day several months) Method of delivery (fermented food, beverages, capsule, tablet) Viability

Probiotic and legislation

EU - EFSA health and nutritional claims USA GRAS generally recognized as save Japan FOSHU Food for specific health use

ISO/IDF standards for probiotic bacteria

ISO 7889:2003 (IDF 117) Enumeration of characteristic organisms in yoghurt ISO 9232:2004 (IDF 146) Yoghurt identification of characteristic organisms ISO 20128:2006 (IDF 192) Presumptive enumeration of Lactobacillus acidophilus in fermented milk products ISO 29981:2010 (IDF 220) Presumptive enumeration of bifidobactaria in fermented milk products

 Research activities:
Probiotics and prebiotics in food and feed Probiotic bacteria in intestinal tract Fermented milk products Probiotic bacteria and human milk

Probiotic bacteria (bifidobacteria) and other bacteria (clostridia) are observed in intestinal and faecal samples: Infant faeces stained by the FISH procedure using bifidobacteria-specific (A) and clostridia-specific (B ) probes

Clostridial and bifidobacterial growth on prebiotics

1,4 1,2 1 0,8 0,6 0,4 0,2 0 Clostridia Bifidobacteria

Ra ffi no se

In ul in

os eP 85

St ac hy os e

La ct ul os e

os eP 95

Ra fti l

Ra fti l

Vi

vin al **

Enumeration of bifidobacteria in fermented milk products

Bifidobacteria are isolated from fermented milk products and identified using phenotypic and molecular methods.

Factors affecting the growth of bifidobacteria in human milk

Vojtech Rada1, Jiri Nevoral2, Eva Vlkov1, Petr Mark3, Jan Sklen4, Sarka Rockova1
1Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences. Prague 6, Kamycka 129. 165 21; 2Pediatric Clinic of Charles University, Prague Motol, Prague 5, V valu 84. 150 06; 3Laboratory of Plant Biotechnologies, Joint Laboratory of Institute of Experimenta Botany Acad. Sci. CR, v.v.i. and Research Institute of Crop Production, v.v.i., Rozvojov 263, 165 02, Prague 6, Czech Republic; 4Departament of Immunology, Institute of Microbiology Acad. Sci. CR, Videnska 1083, Prague, 142 20

Fig. 3: Growth of bifidobacteria in human milk (averages from 15 samples; log CFU/ml)
9 8 7 6 5 4 3 2 1 0

al isA

B. lo ng um

B. lo ng um

B. bi f id um

B. bi f id um

B. bi f id um

B. an im

B. an im

al isB

Fig. 4: Production of lactate in human milk (mg/l)

Fig. 12: Production of lactate and acetate (mM) from HMO

40 35 30 25 20 15 10 5 0 acetate lactate

A B. lo ng um

B. lo ng um B B. an im al isA B. an im al isB

B. bi f id um

B. bi f id um

B. bi f id um

Fig. 8: Cultivation of lysozyme-susceptible B. animalis in human milk: Cells immediately after inoculation (left), destructed cells after 24h (right)

Fig. 7: Cells of lysozyme-resistant B. bifidum after incubation (24h) in human milk

Fig. 1: Lysozyme determination in mothers milk (MM milk sample with lysozyme concentration 32 g/ml)

Fig. 6: Impact of lysozyme on the growth of B. animalis

Fig. 5: Impact of lysozyme on the growth of B. bifidum

Fig. 8: HMO profiles in cultivation media before (standard) and after incubation with bifidobacteria
10,0 ul 0,140 C

0,120 24

Standard B.animalis B B. bifidum B

0,100

0,080

0,060

22

0,040

20

28

30

36

0,020

23

42 min 30,0

-0,010 15,0 20,0 25,0 35,0

Fig. 10: Peak areas of selected HMO before (oligos) and after incubation with bifidobacteria [C*min]
0.045 0.040 0.035 0.030 0.025 0.020 0.015 0.010 0.005 0.000 B. animalis A B. bifidum A B. bifidum C B. longum B B. animalis B B. bifidum B B. longum A oligos 20 22 23 24 28 30 36 42

Fig.11: Cluster analysis based on peak areas of selected HMO[C*min]

Ward`s method Euclidean distances

B. bifidum C B. bifidum B B. bifidum A B. longum A B. animalis B B. animalis A B. longum B oligos

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

Linkage Distance

Conclusions

Probiotics are suitable for youngs Prebiotics are suitable for adults Synbiotics are suitable for both youngs and adults

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