evaluation of probiotics and prebiotics in...
TRANSCRIPT
Department of Microbiology, Nutrition and
Dietetics
prof. Ing. Vojtěch Rada, CSc.
Czech University of Life Sciences Prague
Faculty of Agrobiology, Food and Natural Resources
Evaluation of Probiotics and Prebiotics in Food
Vojtech Rada
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
PROBIOTICS
PREBIOTICS
SYNBIOTICS
PROBIOTICS
„Life microorganisms which when administered in
adequate amounts confer a health benefit on the host“
(FAO/WHO 2002, Lactobacilli, bifidobacteria)
PREBIOTICS
„Nondigestible food ingredients 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) (Inulin, soya
oligosaccharides)
SYNBIOTICS
Combination of probiotics and prebiotics (synergy)
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Intestinal microbiota - FISH
Bifibobacteria
Humans have got approximately 1013 cells of their body.
Humans have got around 1014 bacterial cells in/on their body.
Members of groups such as viruses, fungi and protozoa are also regularly
found in healthy individuals, but form only a minor component of the total
population of resident organisms.
MICROBIOTA - ‘MICROFLORA OF THE BODY’
The microorganisms occur in parts of the body that are exposed to / or
communicate with the external environment (the skin, nose and mouth, and
intestinal and uriogenital tracts etc.).
The majority of bacterial cells is associated with the gastrointestinal
tract.
Internal organs and tissues are normally sterile.
Anaerobic genera
Bifidobacterium
Clostridium
Bacteroides
Eubacterium
Aerobic genera
Escherichia
Enterococcus
Streptococcus
Klebsiellac
MOUTH
ANUS Number of bacterial CFU is caudally increasing
Number of aerobic bacteria is caudally
decreasing
Number of anaerobic bacteria is caudally
increasing
BACTERIAL COLONIZATION OF THE GASTROINTESTINAL
TRACT
CFU = Colony Forming Units
O’Hara and Shanahan, 2006
Protective functions:
Pathogen displacement
Nutrient competition
Receptor competition
Production of anti-
microbial
factors e.g., bacteriocins,
lactic acids
Structural
functions
Barrier fortification
Induction of IgA
Apical tightening of
tight junctions
Immune system
development
Control intra epithelial cell
differentiationand proliferation
Metabolize dietary carcinogens
Synthesize vitamins e.g., biotin,
folate
Ferment non-digestible
dietary residue and endogenous
epithelial-derived mucus
Ion absorption
Metabolic functions:
BENEFITS OF MICROBIOTA FOR THEIR HOST
O’Hara and Shanahan, 2006
Enterotypes: Bacteroides, Prevotella, Ruminococcus
Intestinal microbiota and obesity
Bacteroidetes: Bacteroides, Prevotella
• Firmicutes: Clostridium, Bacillus, Lactobacillus
Changes in faecal flora during life (Mitsuoka, 1992)
Changes in faecal flora during life (Mitsuoka, 1992)
Probiotics Prebiotics
Changes in the composition of intestinal flora during
life (Mitsuoka, 1992) Jakub *22.10.1993 (vaginal delivery, breast-fed)
0
2
4
6
8
10
12
0 50 100 150 200 250
Day after the birth
bac
teri
al
co
un
t in
lo
g C
FU
/g
TC Bif LB E.coli Ent
Kateřina *30.9.1994 (cesarean section, bottle-fed)
0
2
4
6
8
10
12
0 50 100 150 200 250
Day after the birth
Bacte
rial
co
un
ts i
n l
og
CF
U/g
TC Bif LB E. coli Ent
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
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
PROBIOTICS
„Life microorganisms which when administered in
adequate amounts confer a health benefit on the host“
(FAO/WHO 2002, Lactobacilli, bifidobacteria)
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 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
Mechanisms of Probiotics
• Adhesion to intestinal mucus and epithelium
• Aggregation and coaggregation
• Production of antimicrobial substances
• Nutritional effects
• Prevention and treatment of diarrhea
• (Antitumor effects, treatment/prevention of allergic diseases, inflammatory bowel diseases, infection of respiratory tract, etc.)
Nutritional effects
• Alleviation of lactose intolerance symptoms (ß-galactosidase; approved by EFSA)
• Production of vitamins (B, K)
• Reduction of serum cholesterol
Prevention and treatment of diarrhea
• Antibiotic-associated diarrhea (Saccharomyces boulardii)
• Traveler´s diarrhea (prevention)
• Clostridium difficile-associated diarrhea (stool transplantation)
Selection of probiotic bacteria Adherence to epithelial cells (Kmeť, 2004)
Autoaggregation
Bifidobacterium spp.
Clostridium spp.
Coaggregation
Bifidobacterium spp. + Clostridium spp.
Production of antimicrobial substances
• Organic acids
• Hydrogen peroxide
• Bacteriocins (nisin E234, sakacin)
• Reuterin
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, Denmark)
• Bifidobacterium longum BB536 (Murinaga, Japan)
• Bifidobacterium breve (Yakult, Japan)
Commercially available probitic organisms
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
PREBIOTICS
• „Nondigestible food ingredients
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) (Inulin, soya
oligosaccharides)
Criteria for Prebiotics (Roberfroid, 2007)
• 1)resistance to gastric acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal absorption
• 2) fermentation by intestinal microbiota
• 3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being
Prebiotic action→
Prebiotics
• FOS – fructooligosaccharides
• GOS – galactooligosaccharides
• SOS – soya oligosaccharides
• XOS – xylooligosaccharides
• MOS – isomaltooligosaccharides
• HMOs – human milk oligosaccharides
FOS - Fructooligosaccharides
GFn type Fn type
Inulin content in selected plants (Ebringer, 2002)
Plant inulin (FOS) content in g/100g Onion 2 – 7 Garlic 9 – 16 Leek 3 – 10 Sweet potatoes 13 – 20 Jerusalem artychoke 16 – 40 Dandelion 12 – 15 Banana 0.3 – 0.7
FOS – Fructooligosaccharides in
infant fomulas
GOS - Galactooligosaccharides
Produced from whey, lactose
and β-galactosidase
GOS in infant formulas
GOS : FOS = 9 : 1 (8g/L)
SOS – Soya oligosaccharides
• Bifidogenic properties
Soya whey Soya oligosaccharides Protein and salts removing
Raffinose series oligosaccharides (RSO)
raffinose (n = 1)
stachyose (n = 2)
verbascose (n = 3)
SOS – Soya oligosaccharides
SOS (RSO) content in leguminoses (% in dry matter) (Velíšek, 1999)
Leguminose 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 – 0.8 0.02 – 4.8
Human Milk Oligosaccharides (HMOs)
• Neutral - do not contain sialic acid
• Acidic – contain sialic acid
• Main monomers: glucose, galactose, fucose, N-acetylglukosamine and sialic acid
• HMOs: disacharides(lactose), di-,tri-,tetra, penta, …okta-,…dekasaccharides……. (up to date, more then 200 structures identified)
Human milk oligosaccharides
• D-glucose (Glc)
• N-acetylglucosamine (GlcNac)
• Sialic acid (N-acetyl neuraminic acid, Neu 5Ac)
• D-galactose (Gal)
• L-fucose (Fuc)
Basic structure of human milk oligosaccharides
Fucose
Chain
Neu5Ac
Bond
Bond
Tabulka Jednotlivé druhy oligosacharidů jejich struktura a koncentrace v mateřském mléce (Warren et al., 2001) Zkratka Triviální název Struktura Lac Laktóza Galβ(1→4) Glc 2‘-FL 2´-Fukosyllaktóza Fucα(1→2) Galβ(1→4) Glc 3-FL 3-Fukosyllaktóza Galβ(1→4)
Glc Fucα(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) Glc LN/T Lakto-N-neotetraóza Galβ(1→4) GlcNAc β(1→3) Galβ(1→4) Glc LNF-I Lakto-N-fukopentaóza Fucα(1→2) Galβ(1→3) GlcNAc β(1→3) Galβ(1→4) Glc LNF-II Lakto-N-fukopentaóza II Galβ(1→3)
GlcNAc β(1→3) Galβ(1→4) Glc Fucα(1→4) LNF-III Lakto-N-fukopentaóza III Galβ(1→4)
GlcNAc β(1→3) Galβ(1→4) Glc Fucα(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) Glc Galβ(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)
Differences between the human milk and cow's milk (g/l)
Component Human milk Cow milk
Total solids 129 125
Casein 4 28
Albumin 5 7
Lactoferrin 2 0.03
Fat 38 31
Oligosaccharides 8 – 12 0.03 – 0.06
Lysozyme (µg/ml) 400 0.4
Riboflavin 0.43 1.57
Calcium 0.34 1.14
Phosphorus 0.14 0.93
Differences between the human milk and cow's milk (g/l)
Component Human milk Cow milk
Total solids 129 125
Casein 4 28
Albumin 5 7
Lactoferrin 2 0,03
Fat 38 31
Oligosaccharides 8 – 12 0.03 – 0.06
Lysozyme (µg/ml) 400 0.4
Riboflavin 0.43 1.57
Calcium 0.34 1.14
Phosphorus 0.4 0.93
Mechanisms of Human Milk Oligosaccharides
• Prebiotic effects
• Development of central nervous system(sialic acids)
• Prevention of adhesion of pathogens
• Absorption of minerals (Ca, P)
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Synbiotics = combination of probiotics and prebiotics
RSO
FOS
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Guidelines for the evaluation of Probiotics for Food Use (FAO/WHO 2002)
Strian identification by phenotypic and genotypic methods. Genus, species, strain. Deposit strain in international culture collection
Safety assessment. In vitro and/or animal study. Phase 1 human study
Functional characterization. In vitro tests. Animal studies
Double blid, randomized, placebo-controlled (DBPC) phase 2 human trial
Preferable second DBPC
Phase 3, effectiveness trial is appropriate to compare probiotics with standard treatment Probiotic Food
Labeling, content – genus, species, strain designation. Minimum numbers of viable bacteria at end of shelf-life. Proper storage conditions. Corporate contact details for
consumer information.
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Methods for bacterial identification
Method Family genus Species Strain
Phenotypic tests + + +
Mol % G+C + +
Serological tests +
Phagotypization +
FISH + +
Amplificaion of DNA + + +
DNA – DNA Hybridization + +
Genetical fingerprinting + +
Gene sequencing + +
Enzymatic and biochemical methods for bifidobacteria detection and identification
1 2 3 4
Bifidobacteria-positive (1) and bifidobacteria-negative (2) faecal samples (3 – positive
control)
A - bifidobacteria-positive faecal sample, B - bifidobacteria-negative faecal sample (No13 - α-galactosidase, No16 - α-
glucosidase)
fructose-6-phosphate phosphoketolase activity
α-galactosidase and α-glucosidase activity (API ZYM, BioMérieux, Francie)
Biochemical kits API 50 CHL a API ID 32A Rapid (BioMérieux, Francie)
Vlková et al. (2005) J. Microbiol. Meth. 60, 365-373
genus- or species-specific PCR
Nested-DGGE
Molecular methods for detection and identification of intestinal bacteria
FISH procedure using bifidobacteria-specific (A) and clostridia-specific (B) probes
PCR – RAPD
Polyphasic identification
• Combination of morphological, physiological, biochemical, serological, molecular and other methods
General demand (IDF, EU)
• Probiotic fermented milk product should contain 106 CFU/g of probiotic bacteria at the time of sale.
ISO/IDF Standards for Probiotic Bacteria
• ISO 7218 Food Microbiology
• ISO 7889:2003 (IDF 117) Enumeration of yoghurt bacteria
• ISO 9232:2004 (IDF 146) Identification of youghurt bacteria
• ISO 20128:2006 (IDF 192) Enumeration of Lactobacillus acidophilus in fermented milk products
• ISO 29981:2010 (IDF 220) enumeration of bifidobacteria i n fermented milk products
Enumeration of Bifidobacteria
• ISO 29981:2010 (IDF 220)
• TOS medium with mupirocinem (50 mg/L)
• Reliable for milk products
• Not suitable for isolation of bifidobacteria from faecal samples
• Not suitable for the enumeration of B. bifidum species
Products tested
Product
Time to
expiratio
n (days)
Bifidobacteria
l counts
in log CFU.g-1
Mean ± SD
in log CFU.g-1
Species isolated
23 7.17 B.animalis/lactis
17 7.22
17 7.23
16 7.23
ACTIVIA
Yoghurt
10 7.02
7.17 ± 0,09a
15 6.64 B.animalis/lactis
12 6.40 HOLLANDIA
Yoghurt
8 5.89
6.31 ± 0,38a
17 6.42 B.animalis/lactis
12 6.21
OLMA – Dr.
Bif ido
Fermented
milk product 12 6.03
6.22 ± 0,20a
19 6.13 B.animalis/lactis
17 6.07
YOPLAIT
Fermented
milk product 6 5.90
6.03 ± 0,12a
9 5.26 B.animalis/lactis
8 5.46
7 6.00
KEFÍROVÉ
MLÉKO (ABT)
Fermented
milk product 7 5.04
5.44 ± 0,41b
15 2.04 B. longum
11 2.90
9 2.16
17 2.07
RAJO
Yoghurt
8 0
2.37 ± 0,47c
a,b,c
Values with no common superscripts differ (P < 0.05)
Evaluation of Probiotic Food in CZ by Czech Society for Probiotics and Prebiotics
• Enumeration of bacteria
• Identification to the genus and species level
• Tested products: fermented milk and meat products, lyophilized capsules
Bacterial Counts
Product Declared count (logCFU/g)
Measured count
Species identified
Activia (fermented milk drink)
6.00 8.45 ± 0.05 Bifidobacterium animalis subsp. lactis
Activia (youghurt) (Danone)
6.00 8.41 ± 0.28 Bifidobacterium animalis subsp. lactis
Probio fix (S & D Pharma CZ; probiotic capsules)
9.43 10.38 ± 0.12 Bifidobacterium animalis subsp. lactis
Probio fix imun (S & D Pharma CZ; probiotics capsules)
9.43 10.51 ± 0.08 Bifidobacterium animalis subsp. lactis
Selective Enumeration of Bifidobacterium bifidum in probiotic
products
Bifidobacterium bifidum
• Bifidobacterial species specific for human
• Frequent occurence in infant faeces
• Utilize human milk oligosaccharides
• Utilize mucin
• Perspective probiotic species
• In probiotic products usually presented in lyophilized form
Broth for Testing of Mucin Utilization
• Tryptone 5.0 g/ l
• Nutrient broth No. 2 5.0 g/ l
• Yeast extract 2.5 g/ l
• Tween 80 0.5 ml/ l
• Cysteine 0.25 g/ l
• Mucin 20 g/ l
• Bromcresol purpur 0.01 g/l
• pH…………7.4
Tabulka vyuzivani mucinu
Strain Source Mucin utilization
B. adolescentis 1 CCM 4987 -
B. animalis subsp. animalis 1 CCM 4988 -
B. animalis subsp. animalis 2 ATCC 25527 -
B. bifidum 1 Infant faeces +
B. bifidum 2 Infant faeces +
B. bifidum 3 Infant faeces +
B. bifidum 4 Infant faeces +
B. bifidum 5 Infant faeces +
B. bifidum 6 Infant faeces +
B. bifidum 7 Infant faeces +
B. bifidum 8 Adult faeces +
B. bifidum 9 Adult faeces +
B. bifidum 10 Probiotic capsules +
B. bifidum 11 Probiotic capsules +
B. bifidum 12 Probiotic capsules +
B. bifidum 13 Probiotic capsules +
B. breve 1 ATCC 15700 -
B. boum 2 DSMZ 20432 -
B. catenulatum 1 CCM 4989 -
B. choerinum 1 DSMZ 20434 -
B. gallinarum 1 DSMZ 20670 -
B. longum subsp. longum 1 ATCC 15707 -
B. longum subsp. infantis 1 CCM 4990 -
B. merycicum 1 DSZM6482
B. porcinum 1 ATCC 17775 -
B. pseudolongum subsp. globosum 1 DSZM 20092 -
B. pseudolongum subsp. pseudolongum 1 DSZM 20099 -
B. pullorum 1 DSZM 20433 -
B. ruminantium 1 DSZM 6489
B. subtile 1 DSZM 20096 -
B. suis 1 DSZM 20211 -
B. thermacidophilum 1 ATCC 15837 -
B. thermophilum 1 DSZM 20210 -
B. thermophilum 1 DSZM 20212 -
B. bifidum- selective agar
• Tryptone 5.0 g/ l
• Nutrient Broth No. 2, 5.0 g/ l
• Yeast extract 2.5 g/ l
• Tween 80, 0.5 ml/ l
• Cysteine 0.25 g/ l
• Agar10 g/ l
• Mucin 20 g/ l
• Mupirocin 100 mg/l
• pH…………7.4
B. bifidum counts in probiotic products
Product Counts in logCFU/g
Lepicol 8.05±0.02
Biopron 8.10±0.10
Laktobacílky Baby 7.92±0.05
Probioflora 7.17±0.01
AB Active 7.21±0.11
Average 7.69±0.06
Colonies of B. bifidum on MM agar
Identification of B. bifidum
Product F6PPK activity Species-specific
PCR
API 50 CHL +
Rapid ID 32 A
Lepicol + B. bifidum B. bifidum
Probioflora + B.bifidum B. bifidum
Biopron + B.bifidum B. bifidum
Lactobene + B.bifidum B. bifidum
Laktobacílky
Baby
+ B.bifidum B. bifidum
AB Aktiv + B.bifidum B. bifidum
Identification of B. bifidum by MALDI-TOF-MS
Enumeration of L. acidophilus
• ISO 20128:2006 (IDF 192)
• MRS medium with clindamycine (0.1 mg/L)
• Not fully selective
Enumeration of L. casei
• There are no ISO/IDF standard
• MRS agar (Oxoid) with 1.5 % of ox-gall (Oxoid), pH 5.4 (Vinderola a Reinheimer, 2000).
• Rogosa agar(pH 5.4) anaerobic cultivatio at 15 °C for 14 days (Champagne et al., 1997).
• Methods work in fermented milk products but not in fermented meat products
Enumeration of Lactobacillus casei
Enumeration of Lactobacillus casei
Product name Manufacturer
Counts of Lactobacillus casei (milions/1 ml)
1 Actimel - Jahodový Danone 860
2 Actimel - Natur Danone 836
3 Probiotický drink natural Albert quality; Ahold 203
4 Bifidus Natur Pro Viact; Lidl 63
5 Vian Probiotic Drink Hollandia 54
6
Vivacto Probiotic yogurt drink Tesco 52
7 Acti Plus Drink Spar vital 51
8 Probiotic Drink Classic Laktos 6
Identification of Lactobacillus casei to the strain level
Apiweb:
Milblu Natur: L. paracasei ssp. paracasei 99,7 %; 0,79 T
Milblu Jahoda: L. paracasei ssp. paracasei 99,8 %; 0,775 T
Actimel: L. paracasei ssp. paracasei 99,6 %; 0,83 T
Rep-PCR
Vzorek Izolát Poznámka
M
Express DNA
Ladder
(Fermentas)
1 Milblu Natur Kaufland
2 Milblu Natur Kaufland
3 Milblu Natur Kaufland
4 Milblu Jahoda Kaufland
5 Milblu Jahoda Kaufland
6 Milblu Jahoda Kaufland
7 Actimel Danone
8 Actimel Danone
9 Actimel Danone
10 Actimel Danone
11 L. casei 1752
12
L. casei
SHIROTA
13 L. casei
DSM 20011 -
sbírkový
14
L. paracasei
ssp. paracasei
DSM 20312 -
sbírkový
15
L. paracasei
ssp. paracasei
DSM 5622 -
sbírkový
16S rRNA Gene Sequencing
• Danone, Milblu Nature, Milblu Jahoda
• TACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCG
Hsp 6O Gene Sequencing
• Milblu Natur, Milblu Jahoda
• TTATCCGCGAAGGCATGAAGAACGTTACAGCCGGTGCTAATCCTGTTGGCATTCGCACAGGGATTGAAAAGGCAACTAAGGCTGCCGTTGACGAATTGCACAAGATTAGCCACAAAGTTAATGGTAAGAAAGAAATCGCGCAGGTTGCGTCCGTTTCTTCCTCAAATACAGAAGTTGGTAGTCTGATTGCTGACGCCATGGAAAAAGTTGGCCACGATGGTGTGATTACCATTGAAGAAAGCAAAGGGATTGACACTGAACTCTCTGTTGTTGAAGGGATGCAGTTTGATCGCGGCTATCTGAGCCAATACATGGTTACCGATAATGATAAGATGGAAGCTGACCTTGACGATCCTTATATCTTGATCACCGACAAAAAGATTTCCAATATTCAGGACATTTTGCCGCTGTTACAAGAAATCGTTCAACAAGGTAAGGCACTGTTGATCATTGCTGACGACGTTGCTGGTGAAGCATTGCCAACCTTAGTTCTGAACAAGATTCGTGGTACCTTCAATGTTGTTGCGGTTAAGGCCCCCGGCTTCGGCGACCACCATCCTGTGTG
• Danone
• TTATCCGCGAAGGTATGAAGAACGTTACAGCCGGTGCTAATCCTGTTGGCATTCGCACAGGGATTGAAAAAGCAACTAAGGCTGCCGTTGACGAATTGCACAAGATTAGCCACAAAGTTAATGGTAAGAAAGAAATCGCGCAGGTTGCGTCCGTTTCTTCCTCAAATACAGAAGTTGGTAGTCTGATTGCTGACGCCATGGAAAAAGTTGGCCACGATGGTGTGATTACCATTGAAGAAAGCAAAGGGATTGACACTGAACTCTCTGTTGTTGAAGGGATGCAGTTTGATCGCGGCTATCTGAGCCAATACATGGTTACCGATAATGATAAGATGGAAGCTGACCTTGACGATCCTTATATCTTGATCACCGACAAAAAGATTTCCAATATTCAGGACATTTTGCCGCTGTTACAAGAAATCGTTCAACAAGGTAAGGCACTGTTGATCATTGCTGACGACGTTGCTGGTGAAGCATTGCCAACCTTAGTTCTGAACAAGATTCGTGGTACCTTCAATGTTGTTGCGGTTAAGGCCCCCGGCTTCGGCGACCACCATCCTGTGTG
Survival of bifidobacteria in infant formula
Survival of bifidobacteria in infant formula
7.35
7.4
7.45
7.5
7.55
7.6
7.65
0 50 100 150 200 250 300
log KTJ
Počty dnů v měření
Bifidobacterium
Survival of bifidobacteria in infant formula
B. animalis subsp. lactis
B. longum subsp. infantis
Content
• Probiotics, prebiotics – definition
• Intestinal microbiota
• Probiotics – examples, mode of action
• Prebiotics – FOS, GOS, HMOs
• Synbiotics
• Development of new probiotic product
• Identification and enumeration of probiotic bacteria
• Determination of prebiotics in food
Isolation of oligosaccharides from human milk
• Removal of fat – by centrifugation
• Removal of proteins - (precipitation – by ethanol, by
dichloromethane)
• Separation of fractions by gel chromatography
• Analysis of the fractions (TLC - TLC)
• Oligosaccharides were added to the media as the sole
source of sugars for bifidobacteria
Proteins
Oligosaccharides
Lactose
lactose
glucose
Analyses of TLC fractions
(TLC)
Table 1: Strains used and their origin
Strain Source
B. bifidum A infant feaces
B. longum A infant feaces
B. bifidum B infant feaces
B. animalis A fermented milk product A
B. animalis B fermented milk product B
B. longum B lyophilisate
B. bifidum C lyophilisate
• Isolation of strains (TPY agar with mupirocin)
Growth of bifidobacteria in human milk
6,406,58
4,17
7,64
4,73
5,40
7,08 7,14
4
5
6
7
8Počáteční koncentrace
B. bifidum A
B. longum A
B. bifidum B
B. animalis A
B. animalis B
B. longum B
B. bifidum C
Figure 2: Growth of bifidobacteria in human milk (mg/l)
Inoculation
Figure 5: HMO profiles in cultivation media before (standard) and after incubation with bifidobacteria
Figure 6: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
Content of fructans in food
Sample % w/w
Jerusalem artichoke – dried 41.51
Jerusalem artichoke – roasted 31.71
Jerusalem artichoke – red variety 21.70
Jerusalem artichoke – white variety 28.55
Juice from jerusalem artichoke 14.94
Yacon 8.74
Content of raffinose series oligosaccharides in feed and food Feed/Food mM/100 g g/kg*
BR1 3.18±0.34 21.17±2.28
BR2 3.16±0.60 21.06±3.98
BR3 3.33±0.25 22.16±1.66
Soya flour 6.80±1.22 45.33±8.15
Soya flour 8.38±2.20 55.88±14.71
Granulated soya 5.03±1.03 33.56±6.89
Soya cream 2.73±0.49 18.19±3.32
Soya sweet 3.25±0.32 21.62±1.49
Soya bar 4.17±0.80 27.82±5.38
Conclusion
• There is a reliable ISO/IDF method for the enumeration of bifidobacteria in fermented milk product
• Other methods for the enumeration of probiotic bacteria in food need to be developed
• Polyphasic approach is the best way for the identification of probiotic bacteria
Thank you for your attention!