evaluation of probiotics and prebiotics in...

Department of Microbiology, Nutrition and

Dietetics

prof. Ing. Vojtěch Rada, CSc.

[email protected]

Czech University of Life Sciences Prague

Faculty of Agrobiology, Food and Natural Resources

Evaluation of Probiotics and Prebiotics in Food

Vojtech Rada

[email protected]

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





• Synbiotics




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





• Synbiotics




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)


• Bifidobacterium animalis subsp. lactis DN173010 (Danone)

• Bifidobacterium animalis subsp. lactis BB 12 (Chr. Hansen, Denmark)

• Bifidobacterium longum BB536 (Murinaga, Japan)

• Bifidobacterium breve (Yakult, Japan)

Content





• Synbiotics




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





• Synbiotics




Synbiotics = combination of probiotics and prebiotics

RSO

FOS

Content





• Synbiotics




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





• Synbiotics




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


12 6.40 HOLLANDIA

Yoghurt

8 5.89

6.31 ± 0,38a


12 6.21

OLMA – Dr.

Bif ido

Fermented

milk product 12 6.03

6.22 ± 0,20a


17 6.07

YOPLAIT

Fermented

milk product 6 5.90

6.03 ± 0,12a


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)


Probio fix (S & D Pharma CZ; probiotic capsules)


Probio fix imun (S & D Pharma CZ; probiotics capsules)


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 8 Adult faeces +

B. bifidum 9 Adult faeces +

B. bifidum 10 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

http://www.google.cz/imgres?imgurl=http://www.bio-natural.cz/files/big/c5d7984cdbb12436e3f0d8f41f1db091.jpg&imgrefurl=http://www.bio-natural.cz/sortiment87-potravinove-doplnky/714-0-probioflora-probiotikum-s-obsahem-prebioticke-slozky&usg=__GfWeyeVaksT8oERpWnQAtXLfb4s=&h=500&w=367&sz=75&hl=cs&start=26&zoom=1&tbnid=-0B_TYswxR_LIM:&tbnh=123&tbnw=99&ei=6LVmTcUshvqyBt_k2fYM&prev=/images?q=probioflora&hl=cs&sa=G&biw=1362&bih=553&gbv=2&tbs=isch:1&itbs=1&iact=hc&vpx=120&vpy=68&dur=2063&hovh=262&hovw=192&tx=114&ty=121&oei=X7VmTfzGEITXtAbuq8jqDA&page=2&ndsp=27&ved=1t:429,r:0,s:26

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



4 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

http://www.google.cz/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CEIQFjAB&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2045242%2F&ei=BpLmUdv2LKeJ4ASi14D4BA&usg=AFQjCNE9pEszIU6cjv9lz4moGiPA5souTw&sig2=bKIDybG4bOnF_z46n434JQ&bvm=bv.49405654,d.bGE&cad=rja



Hsp 6O Gene Sequencing

• Milblu Natur, Milblu Jahoda

• TTATCCGCGAAGGCATGAAGAACGTTACAGCCGGTGCTAATCCTGTTGGCATTCGCACAGGGATTGAAAAGGCAACTAAGGCTGCCGTTGACGAATTGCACAAGATTAGCCACAAAGTTAATGGTAAGAAAGAAATCGCGCAGGTTGCGTCCGTTTCTTCCTCAAATACAGAAGTTGGTAGTCTGATTGCTGACGCCATGGAAAAAGTTGGCCACGATGGTGTGATTACCATTGAAGAAAGCAAAGGGATTGACACTGAACTCTCTGTTGTTGAAGGGATGCAGTTTGATCGCGGCTATCTGAGCCAATACATGGTTACCGATAATGATAAGATGGAAGCTGACCTTGACGATCCTTATATCTTGATCACCGACAAAAAGATTTCCAATATTCAGGACATTTTGCCGCTGTTACAAGAAATCGTTCAACAAGGTAAGGCACTGTTGATCATTGCTGACGACGTTGCTGGTGAAGCATTGCCAACCTTAGTTCTGAACAAGATTCGTGGTACCTTCAATGTTGTTGCGGTTAAGGCCCCCGGCTTCGGCGACCACCATCCTGTGTG

• Danone

• TTATCCGCGAAGGTATGAAGAACGTTACAGCCGGTGCTAATCCTGTTGGCATTCGCACAGGGATTGAAAAAGCAACTAAGGCTGCCGTTGACGAATTGCACAAGATTAGCCACAAAGTTAATGGTAAGAAAGAAATCGCGCAGGTTGCGTCCGTTTCTTCCTCAAATACAGAAGTTGGTAGTCTGATTGCTGACGCCATGGAAAAAGTTGGCCACGATGGTGTGATTACCATTGAAGAAAGCAAAGGGATTGACACTGAACTCTCTGTTGTTGAAGGGATGCAGTTTGATCGCGGCTATCTGAGCCAATACATGGTTACCGATAATGATAAGATGGAAGCTGACCTTGACGATCCTTATATCTTGATCACCGACAAAAAGATTTCCAATATTCAGGACATTTTGCCGCTGTTACAAGAAATCGTTCAACAAGGTAAGGCACTGTTGATCATTGCTGACGACGTTGCTGGTGAAGCATTGCCAACCTTAGTTCTGAACAAGATTCGTGGTACCTTCAATGTTGTTGCGGTTAAGGCCCCCGGCTTCGGCGACCACCATCCTGTGTG




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

B. animalis subsp. lactis

B. longum subsp. infantis

Content





• Synbiotics




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!

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