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Current Research and Use of Probiotic, Prebiotics and Synbiotics
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Department of Microbiology, Nutrition and
Dietetics
prof. Ing. Vojtěch Rada, [email protected]
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
www.probiotika-prebiotika.cz
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