P ROBI OTI CS and P REBI OTI CS

Bruno Biavati
Dept. of Agro-environmental Sciences and Technologies
Faculty of Agiculture
University of Bologna, Italy

Outlines

Probiotic concept
Probiotics in human
Probiotics in animal
Prebiotic- synbiotic concept
Application of pro-prebiotic in animal feeding

STRESS FACTORS, BAD DIETARY HABITS,

ANTIBIOTIC THERAPIES

breakdown the balance between putative species of protective in favor of

harmful intestinal bacteria, leading to:

Immunity weakening

BENEFICIAL
BACTERIA

Gut disorders
Inflammatory diseases
Infections
Loss of mucosal intestinal integrity
Loss of weight

HARMFUL
BACTERIA

HOW CAN WE ACT to MODULATE GUT MICROBIOTA

and PROMOTE WEEL BEING?

One of the emergence solution is a dietary intervention

intake of beneficial bacteria =

P robiotics

intake of non digestible oligosaccharides =

P rebiotics

intake of both ingredients =

Synbiotics

Probiotics are known to beneficially effect the host:

Balance of the intestinal microbiota
Inflammatory bowel diseases
Alleviation of lactose intolerance
Alleviation of viral/antibiotics associated diarrhoea
Production of vitamins
Improvement of nutrient adsorption
Anticarcinogenic properties
Reduction of cholesterol
Contribution to oral health
Contribution to urogenital health

PROBIOTICS: milestones

from the Greek: pro-bios

He hypothesized that, replacing

or diminishing the number of putrefactive
bacteria in the gut with lactic acid bacteria,
could normalize bowel health and prolong life.
Elie Metchinikoff (1845 1916)

The scientific rationale for the health benefit of lactic acid bacteria
was provided in his book The prolongation of life published in 1907.

At Metchnikoffs time .

Henry Tissier, a French pediatrician, working independently observed that children

with diarrhea had in their stools a low number of bacteria characterized by a peculiar Y shaped
morphology.
These bifid bacteria were, on the contrary, abundant in healthy children. He suggested that
these bacteria could be administered to patients with diarrhoea to help restore a healthy gut
microbiota

PROBIOTICS: milestones

1930s: In Japan

Launch of Yakult (Shirota strain)

1974: Parker

organism s and substances w hich contribute to intestinal m icrobial balance .

1989: Fuller

live m icrobial food/ feed supplem ent w hich beneficially affects the host by
im proving its intestinal m icrobial balance
2002 : FAO/WHO

probiotics are living m icroorganism s w hich upon ingestion in certain

num bers, ex ert health effects, beyond inherent basic nutrition

PROBIOTICS
could be part of the natural microbiota of both animals and humans
Have been used since the beginning of history as starter cultures
Are present in almost all fermented foods-vegetables, meat and dairy products
Have a long history of consumption and safe use

Lactobacillus
spp.

Enterococcus
faecium

Bifidobacterium
spp.

Yeasts

Bacillus spp.

Lactic Acid Bacteria and bifidobacteria are the

best candidates for use as probiotic cultures

THEY HAVE:

The GRAS status

General Recognised as Safe

The QPS status

Qualified Presumption of Safety

Assigned by the
Food and Drug Administration
[FDA]

Assigned by the
European Food Safety Agency
[EFSA]

SAFETY ASSESSMENT
QPS : Qualified Presumption of Safety
Generic risk assessment approach applied by the European Food Safety Authority
(EFSA) to harmonise the assessment of notified biological agents to be used in
food and feed across different EFSAs Scientific Panels and Units

In essence this proposed that a safety assessment of a defined taxonomic group

could be made based on 4 pillars:

Establishing identity
Body of knowledge
End use
Possible pathogenicity

For more information: www.efsa.europa.eu/en/efsajournal/pub/587.htm

Probiotic cultures

- site of activity GI T. Types of action :

S.C. Ng et al., (2009). Mechanisms of Action of Probiotics: Recent Advances

Inflamm. Bowel Dis. 2009;15:300 310

Crosstalk between probiotics and the intestinal mucosa

S.C. Ng et al., (2009). Mechanisms of Action of Probiotics: Recent Advances

Inflamm. Bowel Dis 2009;15:300 310

Key steps for the selection of probiotic strains

Safety and functional Aspects

SAFETY OF PROBIOTICS

7
6
5
4
3
2
1

Clinical trials: colonization and health effects

Modulation of immune response
Adherence to intestinal epithelium

Suppression /reductionof harmful bacteria (antimicrobial

activity)

Resistance to low pH, gastric juice, bile acid and pancreatic juice,
Viability during production and storage

Strain, species and genus safety properties (origin/taxonomic position)

KEYW OR DS TO DEFI N E P R OBI OTI CS

Referred to microorganisms

PROBIOTICS
A NEW WAY FOR THE WELL-BEING

Bioterapeuthic agents
Good benefical bacteria
Health promoting bacteria
Friendly bacteria
Living microbial food ingredient

Prophylactis for intestinal disorders

Referred to food

Lifeway food

Wellness food

Dietary supplements

Functional food

Pharma-food

Food for special health use

HUMAN PROBIOTICS
Most human probiotics are incorporated in foods
especially in dairy products

Also available as food supplemement in the form

of pharmaceutical preparation

ANIMAL PROBIOTICS

Increased use of probiotics as an alternatives to antibiotics

Food safety starts on the farm

Zoonotic pathogens (Salm onella, Cam pylobacter, Escherichia coli, Clostridium ,

Yersinia enterocolitica, Streptococcus aureus, Bacillus cereus ), living in the

gastrointestinal tract of animals, can contaminate meat or milk products during slaughter or
at milking and be transferred to human once a contaminated food, not properly processed,
reaches the consumer. Is important to reduce or eliminate the pathogens at the
origin.

Farm

Slaughterhouse

Processing

Retailers

Consumer

Probiotic cultures in animal feeding

Antibiotics, as growth promoters, were banned in Europe (2006)
Probiotic are alternative to antibiotics and growth promoters
Growing request of organic meat (absence of antibiotic)

When Can Probiotics be Used?

Immediately following birth
to establish a healthy gut microbiota
to prevent establishment of pathogenic bacteria.
Following antibiotic administration
to re-establish beneficial microbiota depleted by antibiotics
to prevent re-infection by pathogens.
To treat or prevent diarrhoea
by reduction or exclusion of pathogenic bacteria including E.coli and Salmonella

Action of probiotics in animals

Improved utilisation of food
by increasing the efficiency of the existing digestive processes & promoting the
digestion of previously indigestible substances.
Reduced intestinal upsets
symptoms include diarrhoea, loss of appetite and poor digestion of food
Improved health
competitive exclusion of pathogen and stimulation of immunity
Establishment/Re-establishment of healthy microbiota
establishment in immature animals
re-establishment following antibiotic use

Dietary modulation of the intestinal microbiota can also be

achieved via oral administration of

P rebiotic com pounds

The prebiotic concept was first proposed
by Gibson and Roberfroid in 1995

A selectively ferm ented ingredient that allow s specific changes,

both in the com position and/ or activity of the gastrointestinal
m icrobiota that confers benefits upon the host w ellbeing and health

To be considered prebiotic, a com pound m ust satisfy a num ber of criteria:

1. it must be resistant to digestion in the upper GIT and therefore resistant to acid
and human enzymatic hydrolysis

*Human gastrointestinal

digestive enzymes are specific for -glycosidic bonds

body lacks the enzymes required to hydrolyze the -links
formed among the units of some monosaccharides
(glucose, galactose, fructose and xylose),

2. It must be a selective substrate for the growth of beneficial bacteria in the colon
(Best candidate are bifidobacteria that produce -fructofuranosidase

3. it must induce luminal or systemic effects that are beneficial to host health

Gibson, G.R.; Roberfroid, M.B. Dietary Modulation of the Human Colonic Microbiota:
Introducing the Concept of Prebiotics. J. Nutr. 1995, 125, 14011412.

Based on the number of monomers linked together

the prebiotics could be classified as:

- Disaccharides
- Oligosaccharides (3-10 monomers)
- Polysaccharides

Most common used prebiotics

Lactulose
Inulin
Fructo-oligosaccharides (FOS)
Galacto-oligosaccharides (GOS)
Transgalacto-oligosaccharides (TOS)

 At present, oligosaccharides are the most common

There is a growing list of candidate prebiotics:

Resistant starch, polydextrose, soybean, isomalto-oligosaccharides, glucooligosaccharides, xylo-oligosaccharides. palatinose, gentiooligosaccharides and
sugar alcohols (such as lactitol, sorbitol and maltitol

HOW PREBIOTICS WORKS ?

OSullivan et al., (2010); Prebiotics from Marine Macroalgae for Human and Animal Health Applications
Mar. Drugs 2010, 8, 2038-2064;

Consumption of a prebiotic compound/food/feed additive

Resistance to digestion
in the upper gastrointestinal tract

Entry to the colon

Selective fermentation by beneficial microbiota

Increased numbers of beneficial bacteria, reduced numbers

of pathogens/putrefactive bacteria

Effects on
bowel function
Production of short
chain fatty acids
Modulation of Immune response
Reduced risk of colon cancer

Increased resistance
to infections

Increased mineral
bioavailability
Effects on Satiety-appetite
Improved gut & bone health
Reduced risk of obesity

Improved growth performance & reduced pathogen shedding

SYNBIOTIC
A MIXTURE OF PROBIOTCS AND PREBIOTICS
THAT BENEFICIALLY AFFECTS THE HOST BY
IMPROVING THE SURVIVAL AND IMPLANTATION
OF LIVE MICROBIAL DIETARY SUPPLEMENTS IN
THE GASTROINTESTINAL TRACT
(Gibson and Roberfroid, 1995)

SYNERGISTIC HEALTH PROMOTING EFFECT

Probiotics and prebiotics application

in animal feeding

EU projects - 6th FRAMEWORK

2004-2009

http://www.qlif.org

2005-2010

www.pathogencombat.com

Weaning modifies the normal gut microbial balance,

leading to increased susceptibility to gut disorders,
infections and diarrhoea.

Implementation of the diet

Probiotics
Prebiotics
Dietary Nitrate

Synbiotics

STRATEGIES TO RESTORE THE INTESTINAL EUBIOSIS

AIM OF THE WORK

Developing and testing new strategies based on probiotics,

prebiotics, synbiotics and dietary nitrate able to modulate the
GIT microbiota, reducing the negative impact of the weaning

WORK PLAN

Probiotics screening
Prebiotics screening

To select the best Synbiotic

Enteric
pathogens

Challenge Trial

Diet rich in Nitrate to prevent pathogens colonization

Best Probiotic AGAINST:

APPLICATION OF THE NEW SYNBIOTIC

probiotic + prebiotic + nitrate

Preliminary trials aimed to select the best feed additive:

In vivo screening of 12 bifidobacteria strains

In vivo assessing the prebiotic effect on endogenous
bifidobacteria
In vivo testing synbiotic preparations

Selection criteria
Ability to increase the number of bifidobacteria in the GIT
Ability to improve the animal growth performance

STUDY DESIGN
Experimental: 3 independent trials
Subjects: 356 pigs weaned at 28 days
Time of trials: 2 weeks
Clinical assessments: growth performance, fever, control of
diarrhoea
Microbiological assessments on samples of caecum contents
through traditional and molecular techniques (qPCR)

Probiotics selection
STRAIN

ORIGIN

SPECIES

DOSE

Su 829

Pig

B. suis

1011 cfu/die

Su 905

Pig

B. suis

1011 cfu/die

Su 932/1

Pig

B. suis

1011 cfu/die

Su 837

Pig

B. choerinum

1011 cfu/die

Su 877

Pig

B. choerinum

1011 cfu/die

Su 891

Pig

B. choerinum

1011 cfu/die

M 354

Yogurt

B. animalis subsp. lactis

1011 cfu/die

Ra 18

Rabbit

B. animalis subsp. lactis

1011 cfu/die

P 32

Chicken

B. animalis subsp. lactis

1011 cfu/die

B 632

Man

B. breve

1011 cfu/die

B 1501

Man

B. breve

1011 cfu/die

B 2501

Man

B. breve

1011 cfu/die

Probiotics Results

Colonization ability was strain specific

2 strains showed the better ability to colonize (108

cfu/gr of caecum content):
a) Ra18 - B. animalis subsp. lactis
b) Su 891 - B. choerinum

Ra18 showed also a positive effect on piglets growth

performance (~ 10%)

SbFOS 1%
10

Prebiotics selection

Ci FOS 1%
10

2 oral doses: 1% - 4% of a normal diet

1. GOS: Galactoolisaccharides from milk whey

2. SbFOS: fructooligosaccharides from sugar beet
3. CiFOS: fructooligosaccharides from cicory inulin

Prebiotics Results
SbFOS at 4% was able to increase the number of
endogenous bifidobacteria
No effect on piglets growth performance

Synbiotic trials
1st

Ra18
107 cfu /die

2nd

Su 891
107 cfu /die

+
SbFOS 4%

+
SbFOS 4%

109 cfu /die

109 cfu /die

+
SbFOS 4%

+
SbFOS 4%

1011 cfu /die

1011 cfu /die

+
SbFOS 4%

+
SbFOS 4%

Synbiotics Results
The recovery of the two probiotic tested was
doses depending
B. anim alis subsp. lactis Ra18 confirmed a
probiotic effect on piglets growth performance

Challenge Trials
I n vivo evaluation of the antimicrobial activity of
the most promising probiotic
B. anim alis subsp. lactis Ra 18

against 2 enteric pathogens:

1. Salm onella enterica serovar typhim urium

2. Enteroxigenic E. coli K88 [ETEC K88]

1. STUDY DESIGN

Subjects: 32 piglets weaned at 28 days

Probiotic supply : 1011cfu
Time of trial: 3 week
Challenge: 1.5 x 109 cfu of S. enterica ser. typhim urium

Results
Presence of Ra18 (~ cfu 108 /gr) in cecum contents
Reduction of Salm onella in cecum contents
Absence of Salm onella in the faeces of the majority
of pigs

2. STUDY DESIGN

Subjects: 32 piglets weaned at 28 days

Probiotic supply: 1011cfu
Time of trials: 3 week
Challenge: 1.5 x 109 cfu of E. coli K88

Results
Increased number of bifidobacteria in the cecum
No reduction on the frequency of diarrhoea

Dietary nitrate
Evaluation

of

the

effect

of

different

nitrate

concentrations
on the stomach and upper intestine microbiota
and the efficacy in controlling Salm onella infection

 Diet rich on nitrates

Additional nitrates from salivar
secretions
Oral bacteria operate the reduction
nitrate to nitrite
NO3
NO2

ORAL CAVITY

STOMACH

INTESTINE

(1)

Partial feed disinfection by low pH (24)

Acid + nitrites: additional
antimicrobial action
First barrier to pathogens

Probiotics
Second barrier

(1) Entero-salivar nitrates circulation through the blood to salivar glands

STUDY DESIGN
4 experimental series
96 piglets weaned at 28 days
2 dietary additions of nitrate: 15 mg/Kg and 150 mg/Kg
Challenge organism: S. enterica ser. typhimurim (1,5 x 109cfu)
Samples: stomach and jejunum contents

HOST RESPONSE
Nitrate addition did not affect daily fed intake and piglets
growth
No effects on the degree of ulcerations
There were low signs of diarrhoea
Reduction of the total microbial count (including
Salmonella) at the dose of 150 mg/Kg

Application in organic breeding

The synbiotic formula
(probiotic + prebiotic + nitrate)
developed on the previous studies was
administered to newly weaned piglets

The synbiotic formula

4% of the sugar beet FOS (SbFOS) Actilight
A dose of nitrate (150 mg/Kg/day)
1011 cfu/day of encapsulated B. animalis subsp. lactis Ra 18

A new technology of microencapsulation was developed by

Probiotical (SME partner within QLIF) and applied to

B. animalis subsp. lactis Ra 18

1. To enhance its survival into the feed

2. To enhance its survival into the stomach

STUDY DESIGN
Experimental series: 3 independent trials
Subjects: 58 piglets weaned at 40 days
Time of trials: 4 weeks
Clinical assessments:

growth performances, presence of diarrhoea or fever

Microbiological assessments: quantification (RT-PCR) of

lactobacilli, bifidobacteria, Bifidobacterium animalis subsp.

lactis, and E. coli on faecal samples collected at:

T0 = starting day experiments
T1 = after 15 days treatment
T2 = after 2 weeks from the end of the treatment

lo g10 CFU/g faeces

Results
12
11
10
9
8
7
6
5
4
3
2
1
0

Bifidobacterium animalis subsp. lactis:

strain Ra 18

Contr ol
Synbiotic

T0

T1

T2

Microencapsulation led to high number in piglet GIT

Persistence after wash out period T2 in all the subjects

Results
Bifidobacterium spp.
12

lo g10 CFU/g faeces

11
10
9
Contr ol
8

Synbiotic

7
6
5
T0

T1

T2

An increase of bifidobacteria
(probiotic and prebiotic effect)

Results

lo g10 gene copies number/g faeces

Lactobacilli
12
11
10
9

Contr ol
Synbiotic

8
7
6
T0

T1

T2

An increase of lactobacilli
(prebiotic effect)

Results
lo g10 gene copies number/g faeces

E. coli
10
9
8
7

Contr ol

Synbiotic

5
4
T0

T1

T2

A reduction of E. coli
(competitive effect)

Results
Wheigts
28
26
24
22

Kg

20
18

Contr ol

16

Synbiotic

14
12
10
8
T0

T1

T2

The synbiotic led to a gain in body wheight

Host Response
The new synbiotic was well tolerated
Piglets remained healthy during all the trials
No cases of diarrhoea
Effect on piglets growth performance

Conclusions
The developed formula showed the potential to
be used to prevent enteric diseases in piglets
without causing any undesirable effect
A positive modulation of the gut microbiota may
also have a significant effect on piglets growth
rate

Use of probiotics and prebiotics:

a strategy to modulate the intestinal microbiota of poultry
and control C. jejuni colonization

Adaptation to the post hatching period

Increased stressors:
feed changes or imbalances
transportation
high stocking densities

may weaken immune functions and thus predispose broilers to

colonization of the gastrointestinal tract by pathogens

 Incidence of human campylobacteriosis during 2008

Reported notification zoonoses rates

in confirmed human cases in EU, 2008
(EFSA, 2010)

Age-specific
distribution
of
the
notification rate of reported confirmed
cases of human campylobacteriosis per
100,000 population in 2008 (EFSA, 2010)

 Incidence of Cam pylobacter spp. in broilers

and fresh poultry meat

Species distribution of
positive samples isolated
from broilers (2008)
20-80% of broilers in
farms positive to

Campylobacter

Species distribution of
Campylobacter isolates
from fresh broiler meat
(2008)
70% positive samples
analyzed

 C. jejuni characteristics and pathogenesis

Campylobacter jejuni

Curved or spiral-shaped cells, Gram-, commensal microorganisms in chicken GIT

Poultry: the major source of human infections no clinical signs in birds
Colonize cecal and cloacal crypts of chickens mainly locates in the mucous layer
Transmission to humans: ingestion of contaminated food or water contact with faecal material
C. jejuni in human: - responsible for human gastroenteritis
- symptoms: abdominal pain, diarrhoea, fever, septicaemia,
- low infective dose

State of the art:

Chicken GIT and Microbiota
Two stomachs: proventriculus and gizzard (pH 1-2)
Rapid rate of passage of digesta (10 h) - Caeca empty:
5-8 times daily
Small intestine: 120 cm
Large intestine:
Two long caecae: ~20 cm
Colon: virtually absent
Mucins differ in structure, folding, glycosylation and charge
Antimicrobial proteins are present at the intestinal
epithelial surface (gallinacins)
Intestinal mucous able to attenuate C. jejuni virulence
Change of the microbiota with ageing

EXPERIMENTAL PART

Probiotics in vitro screening

POULTRY FEEDING TRIALS

Probiotics
Prebiotics

To select the best strains

Antimicrobial activity against pathogens
Survival to GI conditions (pH ,bile salts, starvation)
Survival to food processing (Temp. osmotic stress)
Antibiotic susceptibility (transfer of resistance)

Trial with 2 best strains from selection

Trial with FOS and GOS

SYNBIOTIC product

W
O
R
K
P
L
A
N

I n vivo trial: STUDY DESIGN

Grouped in collective boxes
Ad libitum feed and water
Monitoring of environmental conditions and animal weight
Sampling (10 chickens sterile condition):
T0 before administration
T1 after 15-day administration
T2 7 days after stopping administration

Faecal samples immediately

transferred to the LAB

MOLECULAR ANALYSIS

Analysis Workflow:
Culture-dependent
techniques

Faecal samples

Microbiota analysis

to assess
microorganisms
vitality in faeces

Culture-independent
technique
DNA extraction

Protocols optimization

qPCR analysis

SybrGreen Chemistry

Targets

Bifidobacterium spp.
Bifidobacterium longum
Lactobacillus spp.
Lactobacillus plantarum
Campylobacter spp.
C. jejuni

StepOneTM
Real-Time PCR SYSTEM

RESULTS:

PROBIOTIC trial:
Probiotic administration
(frozen cultures ~108 cfu

B. longum PCB133
L. plantarum PCS20

p<0.01

* ANOVA analysis with GLM procedure of SAS

p<0.05

PREBIOTIC trial:
Prebiotic
administration

Fructo-oligosaccharides FOS
(Actilight) 0.5 %
Galacto-oligosaccharides GOS (CUP
Oligo P) 3%

p<0.05

p<0.05

Increase of bifidobacteria (p<0.05)

GOS treated group

 Preliminary conclusions:
Lactobacillus plantarum was not detected in faecal samples
Bifidobacterium longum showed persistence
Decrease of C. jejuni

Increase of bifidobacteria (p<0.05)

GOS treated group

No significant weight variation between treated groups

OUR

CHOICE

B. longum PCB133 + GOS

Evaluation of the survival of the microencapsulated probiotics

12

PCS20
PCB133

Vitality (log CFU/g)

11
10

Microbac PCS & PCB : > 1 x 109 cfu/g

(Probiotical S.p.A.)

9
8

Room temperature

T0

6
5

Microencapsulated products:

10

20

30

40

50

60

T7

T14

T30

T60

PCS20 CFU/g

11,3

11,5

10,7

10,5

9,6

PCB133 CFU/g

10,4

9,5

9,5

9,5

8,9

Time (days)

MixedPCS
MixedPCB

12

Feed:Microbac = 50:50

Vitality (log CFU/g)

11

Room temperature

10
9
8

T0

T7

T14

T30

T60

Mixed PCS CFU/g

11,6

11,6

10,3

10,1

8,6

Mixed PCB CFU/g

8,9

8,8

8,6

8,2

7,6

7
6
5

10

20

30

Time (days)

40

50

60

SYNBIOTIC trial (using the microencapsulated added to feed)

SYN: GOS + PCB133

p<<0.01

Final Conclusions:

Microencapsulation technology ensure a better survival of the probiotic microorganisms

during gastric transit

B. longum PCB133 showed good persistence

Improved uniformity of chicken microbiota

C. jejuni level can be decreased through synbiotic additives

Several countries are implementing strategy to reduce Campylobacter contaminated

flocks (The use of probiotic and prebiotic prooved to be a good stategy)

Questions?