PROBIOTICS and PREBIOTICS

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

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

breakdown the balance between putative species of protective in favor of

harmful intestinal bacteria, leading to:

HARMFUL BACTERIA

BENEFICIAL BACTERIA

HOW CAN WE ACT to MODULATE GUT MICROBIOTA

and PROMOTE WEEL BEING?

One of the emergence solution is a dietary intervention

intake of non digestible oligosaccharides = Prebiotics

intake of beneficial bacteria = Probiotics

intake of both ingredients = Synbiotics

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 are known to beneficially effect the host:

Elie Metchinikoff (1845 1916)

PROBIOTICS: milestones

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.

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

from the Greek: pro-bios

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

1930s: In Japan Launch of Yakult (Shirota strain)

1974: Parker organisms and substances which contribute to intestinal microbial balance. 1989: Fuller

live microbial food/ feed supplement which beneficially affects the host by improving its intestinal microbial balance 2002 : FAO/WHO

probiotics are living microorganisms which upon ingestion in certain

numbers, exert health effects, beyond inherent basic nutrition

PROBIOTICS: milestones

8

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.

Bifidobacterium spp.

Yeasts

Enterococcus faecium

Bacillus spp.

PROBIOTICS

THEY HAVE:

Lactic Acid Bacteria and bifidobacteria are the best candidates for use as probiotic cultures

The GRAS status General Recognised as Safe

Assigned by the Food and Drug Administration

[FDA]

The QPS status Qualified Presumption of Safety

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 GIT. Types of action :

S.C. Ng et al., (2009). Mechanisms of Action of Probiotics: Recent Advances Inflamm. Bowel Dis. 2009;15:300 310

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

Key steps for the selection of probiotic strains

Safety and functional Aspects

Strain, species and genus safety properties (origin/taxonomic position) 1

2 Viability during production and storage

3 Resistance to low pH, gastric juice, bile acid and pancreatic juice,

4 Suppression /reductionof harmful bacteria (antimicrobial activity)

5 Adherence to intestinal epithelium

6 Modulation of immune response

7 Clinical trials: colonization and health effects

SAFETY OF PROBIOTICS

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

Prophylactis for intestinal disorders

KEYWORDS TO DEFINE PROBIOTICS

Referred to food Lifeway food Wellness food Dietary supplements Functional food Pharma-food Food for special health use

PROBIOTICS A NEW WAY FOR THE WELL-BEING

Most human probiotics are incorporated in foods especially in dairy products

Also available as food supplemement in the form of pharmaceutical preparation

HUMAN PROBIOTICS

Increased use of probiotics as an alternatives to antibiotics

ANIMAL PROBIOTICS

Farm Slaughterhouse Processing Retailers Consumer

Zoonotic pathogens (Salmonella, Campylobacter, 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.

Food safety starts on the farm

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

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

Action of probiotics in animals

Dietary modulation of the intestinal microbiota can also be achieved via oral administration of

Prebiotic compounds

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

A selectively fermented ingredient that allows specific changes,

both in the composition and/ or activity of the gastrointestinal

microbiota that confers benefits upon the host wellbeing and health

To be considered prebiotic, a compound must satisfy a number 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.

Most common used prebiotics

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

Based on the number of monomers linked together the prebiotics could be classified as:

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

At present, oligosaccharides are the most common

There is a growing list of candidate prebiotics: Resistant starch, polydextrose, soybean, isomalto-oligosaccharides, gluco-

oligosaccharides, xylo-oligosaccharides. palatinose, gentiooligosaccharides and

sugar alcohols (such as lactitol, sorbitol and maltitol

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

HOW PREBIOTICS WORKS ?

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

Production of short chain fatty acids

Effects on bowel function

Increased resistance to infections Increased mineral

bioavailability

Effects on Satiety-appetite

Modulation of Immune response

Reduced risk of colon cancer

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

http://www.qlif.org www.pathogencombat.com

2005-2010 2004-2009

EU projects - 6th FRAMEWORK

Weaning modifies the normal gut microbial balance,

leading to increased susceptibility to gut disorders,

infections and diarrhoea.

Probiotics Prebiotics Dietary Nitrate

Synbiotics

STRATEGIES TO RESTORE THE INTESTINAL EUBIOSIS

Implementation of the diet

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

APPLICATION OF THE NEW SYNBIOTIC probiotic + prebiotic + nitrate

4

Probiotics screening Prebiotics screening To select the best Synbiotic 1

2 Challenge Trial Best Probiotic AGAINST: Enteric pathogens

3 Diet rich in Nitrate to prevent pathogens colonization

WORK PLAN

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%)

Prebiotics selection

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

Ci FOS 1%Ci FOS 1%10 10

SbFOSSbFOS 1%1%10 10

Ci FOS 1%Ci FOS 1%10 10

SbFOSSbFOS 1%1%10 10

Ci FOS 1%Ci FOS 1%10 10

SbFOSSbFOS 1%1%10 10

Ci FOS 1%Ci FOS 1%10 10

SbFOSSbFOS 1%1%10 10

Prebiotics Results

SbFOS at 4% was able to increase the number of endogenous bifidobacteria

No effect on piglets growth performance

Ra18

1011 cfu /die +

SbFOS 4%

109 cfu /die +

SbFOS 4%

107 cfu /die +

SbFOS 4%

1011 cfu /die +

SbFOS 4%

109 cfu /die +

SbFOS 4%

107 cfu /die +

SbFOS 4%

Su 891

Synbiotic trials

1st 2nd

Synbiotics Results

The recovery of the two probiotic tested was doses depending

B. animalis subsp. lactis Ra18 confirmed a probiotic effect on piglets growth performance

Challenge Trials

In vivo evaluation of the antimicrobial activity of the most promising probiotic

B. animalis subsp. lactis Ra 18

against 2 enteric pathogens:

1. Salmonella enterica serovar typhimurium 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. typhimurium

Results

Presence of Ra18 (~ cfu 108 /gr) in cecum contents

Reduction of Salmonella in cecum contents Absence of Salmonella 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 Salmonella infection

ORAL CAVITY

STOMACH

INTESTINE

Diet rich on nitrates Additional nitrates from salivar

secretions Oral bacteria operate the reduction

nitrate to nitrite NO3 NO2

Partial feed disinfection by low pH (2- 4)

Acid + nitrites: additional antimicrobial action

First barrier to pathogens

Probiotics Second barrier

(1)

(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

Results

Microencapsulation led to high number in piglet GIT

Persistence after wash out period T2 in all the subjects

0123456789

101112

T0 T1 T2

log 1

0C

FU/g

faec

es

Bifidobacterium animalis subsp. lactis: strain Ra 18

Control

Synbiotic

56

7

8

9

10

11

12

T0 T1 T2

log 1

0C

FU/g

faec

es

Bifidobacteriumspp.

Control

Synbiotic

Results

An increase of bifidobacteria

(probiotic and prebiotic effect)

Results

An increase of lactobacilli

(prebiotic effect)

6

7

8

9

10

11

12

T0 T1 T2

log 1

0ge

ne c

opie

s nu

mbe

r/g

faec

es

Lactobacilli

Control

Synbiotic

Results

A reduction of E. coli

(competitive effect)

4

5

6

7

8

9

10

T0 T1 T2

log 1

0ge

ne c

opie

s nu

mbe

r/g

faec

esE. coli

Control

Synbiotic

Results

The synbiotic led to a gain in body wheight

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10

12

14

16

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22

24

26

28

T0 T1 T2

Kg

Wheigts

Control

Synbiotic

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

may weaken immune functions and thus predispose broilers to

colonization of the gastrointestinal tract by pathogens

Adaptation to the post hatching period

Increased stressors:

feed changes or imbalances transportation high stocking densities

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 human campylobacteriosis during 2008

Incidence of Campylobacter 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

Rapid rate of passage of digesta (10 h) - Caeca empty: 5-8 times daily

Two stomachs: proventriculus and gizzard (pH 1-2)

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

State of the art: Chicken GIT and Microbiota

Change of the microbiota with ageing

Probiotics in vitro screening To select the best strains 1

2 Probiotics Prebiotics

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

POULTRY FEEDING TRIALS

Trial with 2 best strains from selection Trial with FOS and GOS

SYNBIOTIC product

W O R K P L A N

EXPERIMENTAL PART

In 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

DNA extraction

Microbiota analysis Culture-dependent techniques

Culture-independent technique

Analysis Workflow:

qPCR analysis

to assess microorganisms

vitality in faeces

Faecal samples

Protocols optimization

Targets

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

SybrGreen Chemistry

StepOneTM

Real-Time PCR SYSTEM

RESULTS: PROBIOTIC trial:

p

p

Prebiotic administration

Fructo-oligosaccharides FOS (Actilight) 0.5 %

Galacto-oligosaccharides GOS (CUP Oligo P) 3%

PREBIOTIC trial:

p

No significant weight variation between treated groups

Increase of bifidobacteria (p

T0 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

Feed:Microbac = 50:50

Room temperature

Room temperature

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

(Probiotical S.p.A.)

Microencapsulated products:

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

0 10 20 30 40 50 605

6

7

8

9

10

11

12

Vitali

ty (lo

g CFU

/g)

Time (days)

PCS20 PCB133

0 10 20 30 40 50 605

6

7

8

9

10

11

12

Vitali

ty (lo

g CFU

/g)

Time (days)

MixedPCS MixedPCB

Evaluation of the survival of the microencapsulated probiotics

SYNBIOTIC trial (using the microencapsulated added to feed)

p

Final Conclusions:

Microencapsulation technology ensure a better survival of the probiotic microorganisms during gastric transit

B. longum PCB133 showed good persistence

C. jejuni level can be decreased through synbiotic additives

Improved uniformity of chicken microbiota

Several countries are implementing strategy to reduce Campylobacter contaminated flocks (The use of probiotic and prebiotic prooved to be a good stategy)

Questions?

PROBIOTICS and PREBIOTICSBruno BiavatiDept. of Agro-environmental Sciences and TechnologiesFaculty of AgicultureUniversity of Bologna, ItalySlide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9SAFETY ASSESSMENTSlide Number 11Slide Number 12Slide Number 13KEYWORDS TO DEFINE PROBIOTICSSlide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27AIM OF THE WORKSlide Number 29Preliminary trials aimed to select the best feed additive:Selection criteriaSTUDY DESIGNProbiotics selectionProbiotics ResultsPrebiotics selectionPrebiotics ResultsSlide Number 37Synbiotics ResultsChallenge Trials1. STUDY DESIGNResults2. STUDY DESIGNResultsDietary nitrateSlide Number 45STUDY DESIGNHOST RESPONSEApplication in organic breedingThe synbiotic formulaSlide Number 50STUDY DESIGNResultsResultsResultsResultsResultsHost ResponseConclusionsSlide Number 59Slide Number 60Slide Number 61Slide Number 62Slide Number 63Slide Number 64Slide Number 65Slide Number 66Slide Number 67Slide Number 68Slide Number 69Slide Number 70Slide Number 71Slide Number 72Slide Number 73Slide Number 74Slide Number 75