Department of Microbiology, Nutrition and

Dietetics

prof. Ing. Vojtěch Rada, CSc.rada@af.czu.cz

Czech University of Life Sciences Prague

Faculty of Agrobiology, Food and Natural Resources

Current Research and Use of Probiotic, Prebiotics and Synbiotics

Prof. Ing. Vojtěch Rada, CSc.

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)

Newborn

Full acces to mother

Complete

microflora

Protection

Limited acces to

mother

Incomplete protection Deficient

microflora

+ Probiotics

Protective role of probiotics (Fuller, 1989)

Probiotic bacteria

Lactic acid bacteriaLactobacillus acidophilus

Lactobacillus casei

Lactobacillus rhamnosus

Lactobacillus salivarius

Lactobacillus plantarum

Lactobacillus delbrueckii ssp.

bulgaricus

Lactococcus lactis

Enterococcus faecium

Streptococcus thermophilus

Pediococcus pentosaceus

BifidobacteriaBifidobacterium animalis ssp. lactis

Bifidobacterium longum

Bifidobacterium bifidum

Bifidobacterium breve

Bifidobacterium infantis

Bifidobacterium pseudolongum

Bifidobacterium thermophilum

Other bacteriaEscherichia coli

Bacillus sp.

Clostridium butyricum

FungiSaccharomyces 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.

CoaggregationBifidobacterium 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 in g/100gOnion 2 – 7Garlic 9 – 16Leek 3 – 10Chicory 30 – 47Sweet potatoes (Tuberus) 16 – 20Dandelion 12 – 15Banana 0,3 – 0,7

FOS (oligofructose) – Fructooligosaccharides

in infant formulas

GOS - Galactooligosaccharides

GOS in infant formulas

GOS/FOS = 9:1

Raffinose series oligosaccharides (RSO)

raffinose (n = 1)

stachyose (n = 2)

verbascose (n = 3)

SOS – Soya oligosaccharides

Stachyose and raffinose content in leguminoses (in % of dry matter; Velíšek, 1999)

Plant Raffinose Stachyose

Bean 0.3 – 1.1 3.5 – 5.6

Pea 0.6 – 1.0 1.9 – 2.7

Lentil 0.3 – 0.5 1.9 - 3.1

Soya 0.2 – 1.8 0.02 – 4.8

Monomers of human milk oligosacharides

• D-glucose (Glc)

• N-acetylglucosamine (GlcNac)

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

• D-galactose (Gal)

• L-fucose (Fuc)

Basic structure of HMO

Fucose

Řetězec

Neu5Ac

Vazba

Vazba

Tabulka Jednotlivé druhy oligosacharidů jejich struktura a koncentrace v mateřském mléce (Warren et al., 2001)Zkratka Triviální název StrukturaLac Laktóza Galβ(1→4) Glc2‘-FL 2´-Fukosyllaktóza Fucα(1→2) Galβ(1→4) Glc3-FL 3-Fukosyllaktóza Galβ(1→4)

GlcFucα(1→3)

LDFT Laktodifukotetraóza Fucα(1→2) Galβ(1→4)

Glc

Fucα(1→3)LNT Lakto-N-tetraóza Galβ(1→3) GlcNAc β(1→3) Galβ(1→4) GlcLN/T Lakto-N-neotetraóza Galβ(1→4) GlcNAc β(1→3) Galβ(1→4) GlcLNF-I Lakto-N-fukopentaóza Fucα(1→2) Galβ(1→3) GlcNAc β(1→3) Galβ(1→4) GlcLNF-II Lakto-N-fukopentaóza II Galβ(1→3)

GlcNAc β(1→3) Galβ(1→4) GlcFucα(1→4)

LNF-III Lakto-N-fukopentaóza III Galβ(1→4)

GlcNAc β(1→3) Galβ(1→4) GlcFucα(1→3)

LDFH-I Lakto-N-difukohexaóza I Fucα(1→2) Galβ(1→3)

GlcNAc β(1→3) Galβ(1→4) Glc

Fucα(1→4)LDFH-II Lakto-N-difukohexaóza II Galβ(1→3)

GlcNAc β(1→3) Galβ(1→4)Fucα(1→4)

Glc

Fucα(1→3)MFLNH-III Monofucosyllakto-N-hexaóza III Fucα(1→3)

GlcNAc β(1→6) Galβ(1→4) Galβ(1→4) GlcGalβ(1→3)GlcNAc β(1→3) DFLNHa Difukosyllakto-N-hexaóza a Fucα(1→3)

GlcNAc β(1→6)Galβ(1→4) Galβ(1→4) Glc

Fucα(1→2) Galβ(1→3) GlcNAc β(1→3)

Gorilla gorilla

Pongo abelii

Pan troglodytes

Pan pansicuss Pan pansicus

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

• Martín R., Langa S., Reviriego C., Jimínez E., Marín M.L., Xaus J., Fernández L., Rodríguez 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

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Raffi

nose

Sta

chyo

se

Lactulos

e

Inulin

Rafti

lose

P85

Rafti

lose

P95

Vivinal**

Clostridia

Bifidobacteria

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 Vlková1, Petr Maršík3, Jan Sklenář4, 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 Úvalu 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., Rozvojová

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)

0

1

2

3

4

5

6

7

8

9

B.b

ifidu

mA

B.b

ifidu

mB

B.b

ifidu

mC

B.lo

ngum

A

B.lo

ngum

B

B.a

nim

alisA

B.a

nim

alisB

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

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

0

5

10

15

20

25

30

35

40

B.b

ifidu

mA

B.b

ifidu

mB

B.b

ifidu

mC

B.lo

ngum

A

B.lo

ngum

B

B.a

nim

alisA

B.a

nim

alisB

acetate

lactate

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 withlysozyme concentration 32 µg/ml)

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

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

15,0 20,0 25,0 30,0 35,0

min

10,0 ul

20

22

23

24

28

30 36

42

-0,010

0,020

0,040

0,060

0,080

0,100

0,120

0,140µC

Standard

B.animalis B

B. bifidum B

Fig. 8: HMO profiles in cultivation media before (standard) and after

incubation with bifidobacteria

Fig. 10: Peak areas of selected HMO before (oligos) and after incubation with bifidobacteria [µC*min]

20

22

23

24

28

30

36

42B. animalis A

B. animalis B

B. bifidum A

B. bifidum B

B. bifidum C

B. longum A

B. longum B

oligos

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

Ward`s method

Euclidean distances

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

Linkage Distance

oligos

B. longum B

B. animalis A

B. animalis B

B. longum A

B. bifidum A

B. bifidum B

B. bifidum C

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

Conclusions

• Probiotics are suitable for youngs

• Prebiotics are suitable for adults

• Synbiotics are suitable for both youngs and adults

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