BIBLIOGRAPHY

Animal life wouldnt be possible without

mibrobes in the digestive tract
Louis Pasteur

This review explores an invisible and fascinating

world, a protective world essential for life: the world
of microorganisms, which live in our environment
includingourbodies(skin, mucosa, digestivetract,).
This is the story of a probiotic live yeast for Humans
andother animals: the yeast Saccharomyces cerevisiae
boulardii.
Thisreviewprovidesthescientificbackgroundwhich
made it possible to establish the precise modes of
action of the probiotic yeast Saccharomyces cerevisiae
boulardii.
The probiotic Saccharomyces cerevisiae boulardii
provides benefits in terms of health, nutrition and
safety to livestock producers. It contributes to the
improvement in breeding, nutrition, health and
housing as well as overall agriculture in the 21st
century.

IMPACT OF THE USE OF

Saccharomyces cerevisiae boulardii
IN MONOGASTRICS

SUPPORT FOR ALL PROFESSIONNALS

OF NUTRITION AND ANIMAL HEALTH

www.lallemand.com
January 2004

Impact of the use of

Saccharomyces cerevisiae boulardii

in monogastrics

AUTHORS
Julien Delforge

Dr Tom Thompkins

(Monogastric technical service Lallemand,

France)

(Scientific manager Institut Rosell,

Lallemand, Canada)

Dr Frdrique Chaucheyras
Durand

(Technical manager Lallemand, France)

(INRA, France)

Eric Chevaux
Bruno Rochet

Dr Henri Durand
(Scientific manager Lallemand, France)

Dr Mark Franklin
(Microbiologist, Lallemand North America)

(Development manager Lallemand, France)

Laurent Dussert
(Probiotic manager Lallemand, France)

VALIDATION COMMITTEE
Michael Yaeger DVM, PhD, DACVP

Prof Claude Chevrier

Vet diagnostic Laboratory

College of Veterinary Medecine
Iowa State University, USA

Dpartment of Animal Production

University of Tours, France

Prof Jean Yves Jouglar

National Vet College of Toulouse
President of French Association of Porcine Veterinary Medecine

Introduction
Yeast: A Human Conquest
An Historical Utilization
A Natural Production

p. 11

The Porcine Gastrointestinal Ecosystem

Concept of the Rearing Environment
The Digestive Flora of the Piglet
Complexity and Function of the Digestive Flora
Sequence of Microbial Establishment

p. 17

The Imbalance
Origin of the Imbalance
Solutions
antibiotics
probiotics

p. 23

The rearing environment represents an evolving ecosystem in animals, man, and

microorganisms. These microorganisms interact with other living entities in the
environment.Theyarepresentinecologicalnichesandrepresent,atcertainlevels,
a vital and particularly original ecosystem. The microbial ecosystem exerts a
criticalhealthcontrolduringtherearingperiod.Thenormaldevelopmentofthe
digestivesysteminthepigprovidesforprotectionfrommicrobialpathogens.

Saccharomyces cerevisiae boulardii

Origin of S. c. boulardii
Unique Taxonomy and Specificity
The Shield Effect of Saccharomyces cerevisiae boulardii
effect against Clostridium difficile toxins
adherence of Certain pathogens by
Saccharomyces cerevisiae boulardii
stimulating effect on intestinal mucosa

p. 35

Conclusion

p. 38

Bibliography

p. 42

Associated articles
4

The digestive microflora of the pig constitutes a complex microbial ecosystem.

Equilibrium is fundamental for protection against intestinal problems and
determines future growth of the animal. A stable flora plays a role in
establishment of a barrier to hinder the implantation of certain microbial
pathogens (Nurmi,1973).Disturbancesfrombothinternal(growth,weaning)and
external (nutrition, transport) sources provoke an unbalance in the microbial
populations(Tournut,1979).
Typical production practices have used antibiotics to control pathogens. They
have been used for decades in animal production and often without proof of
efficacy against the disease in question. Two applications exist. They are:
therapeuticusage for treatment or prevention of a pathogenic animal disease and
systematic subtherapeutic use for growth promotion in the absence of a disease
condition.
This subtherapeutic usage has drawn public attention from the World Health
Organization (WHO) due to the possible risk of the development of bacterial
resistance (European Commission Report, March 25, 2002). From this the
PrecautionaryPrinciplehasevolvedwhichstatesthattheuseof allantibioticsfor
growthpromotionwillceasebyJanuary1st,2006.

Introduction

SUMMARY
p. 5
p. 7

Introduction

Consumers are now more aware of the image, quality, and origin of the food
they consume. They refuse to tolerate the systematic usage of antibiotics in
production practices. These events dictate worldwide animal production to
rationalizeantibioticusageandcallforthedevelopmentofnew solutions.
One of these solutions, the use of probiotics, has received favorable opinions
fromboththeconsumerandthelegislature.Thisapproachworkstobalancethe
direct environment of the animal, notably the microenvironment that constitutes
thedigestivefloraoftheanimal.
This novel concept has received overwhelming approval in the use of lactic acid
bacteria in yogurt cultures. The utilization of live yeast, Saccharomyces cerevisiae
boulardii (S. c. boulardii), in man is accepted by health officials in a number of
differentcountriesaroundtheworld.
Inswineproduction,numerouspositivestudieswithS.c.boulardii haveresultedin
EuropeanandCanadianregistrationasafeedadditiveforimprovedperformance.
Saccharomycescerevisiaeboulardii iscommerciallyavailableasLevucellSB,andhas
beenproducedsince1993andutilizedinmanyareasoftheworld includingNorth
America,Brazil,Africa,theOrient,andEurope.

Yeast : A human conquest

- Microorganisms represent the premier form of life on the
planet.
- Microorganisms
processes.

play

central

role

biological

- The first observations of microorganisms were made in

1674 by a Dutch scientist.
- Microorganisms have been exploited to modify food in
many ways (baking, beer, wine, cheese).
- The production of yeast is a natural process.

in

Yeast : a Human Conquest

Introduction

Yeast : a Human Conquest

1. Yeast : a human conquest

1.1 An historical utilization

. Another revolutionary usage is the use of yeast in winemaking. It represents an

ancient alcoholic beverage. Ancient Hebrews cultivated vineyards. The Greeks and
Romans also evolved the art of wine production. Religious celebrations, weddings,
tributes; wine has played an important role in the events of mankind. Throughout
history, the involvement of yeast in wine production wasnt well understood until
Pasteurs discovery of yeast. Through his discovery, much scientific progress has
been made.

Discovery of microorganisms

Health

In 1674 the Dutch scientist Antoine Leeuwenhoek made the first observations of
microorganisms through a microscope of his design. Since then, man has
discovered microorganisms virtually everywhere. They are the true colonizers of the
world. Several hundred species of microorganisms coexist with man (bacteria,
yeast). They can be recovered from soil, air, skin, mouth, digestive tract Almost
all higher life forms depend on microorganisms and their processes for life.
Among the microorganisms, yeast are probably the most studied. Through the
microscope, they appear as round or ovoid cells. For biologists yeast are classified
above other single-celled organisms because they combine properties of bacteria
(speed of replication and simplicity of their nutritional requirements) along with
properties found in higher life forms.
Without prior knowledge, man consumed yeast for eons in food. Today he accepts
this concept of ingesting living yeast organisms as beneficial because they enrich his
food and contribute to his overall health. These microorganisms are vital because
they play a central role in biological processes.

Humans have historically used yeast products as health aids. Yeast has been
used for 5 000 to 8 000 years when Babylonians, Egyptians, and Celts, who
also used it for alcohol production, used yeast for benefits on the skin and color.

Interest of the yeast

Of the microorganisms, yeast is fundamentally the most diverse. The empirical
utilization and peaceful existence with man has existed for centuries.

Around 370 BC, Hippocrates discovered the diuretic action of yeast and
considered it a drug.
During the middle ages, clergy used yeast against leprosy to prevent
contamination. It was also used to cure rubella and scarlet fever.
At the start of the 20th century, Indochinese used a Native Indonesian cure for
diarrhea by drinking tea made with tropical fruits (lychee and mango). It has since
been discovered that the agent in the tea responsible for stopping diarrhea was a
yeast (Saccharomyces cerevisiae boulardii).

Nutritional source
Yeast are a superior nutritional source of the edible fungi: they are high in protein
(45 to 50%), and B vitamins (B1, B2, B12). Taken in sufficient quantity, they can
prevent protein and vitamin malnutrition.

Alimentary transformation
This organism has been exploited to modify food in many ways (baking, beer, wine,
cheese).
. Egyptians used yeast in bread making 3000 years ago. Since the history of baking
bread began, yeast has been an important partner. Bread, recognized around the
world for its nutritional value and commonality in most cultures, relies principally
on the use of yeast.

Yeast : a Human Conquest

Yeast : a Human Conquest

1.2 Yeast : a natural production

The production of yeast is a natural process. The main raw material, molasses,
comes from the cane and sugar beet industries. Molasses contains 60% fermentable
sugars (predominantly sucrose) on a dry matter basis. The remaining portion is
used for its nutritional value in food. In addition to molasses; water, vitamins,
minerals and trace elements are added in a continuous flow with air to ensure the
optimal production of yeast cells.

The Porcine Gastrointestinal ecosystem

- The rearing environment can be considered as an open universe
where different ecosystems integrate
- The balance of the intestinal flora determines animal health
and performance.
- The digestive ecosystem constitutes 400 to 500 different species
of microorganisms; feces contains 1011 CFU (Colony Forming
Units) of microorganisms per gram.
- These microorganisms can have positive or negative actions, as
a function of their concentrations.
- The digestive flora of the pig begins to develop at birth; the goal
is then to maintain a balance.

Figure 1 : Schematic representation of yeast production

10

11

The Porcine gastrointestinal ecosystem

2. The Porcine Gastrointestinal Ecosystem

2.1 Concept of the Rearing Environment
Raibaud defines an ecosystem as the set of all living populations as well as all of the
inert components that interact in a region defined of space and time (Raibaud and
Ducluzeau, 1980). The rearing environment can be considered as an open universe
where different ecosystems integrate. Different visible ecosystems coexist: the
building, the room, the pen, and the animals. Within these ecosystems, microecosystems also exist, often invisible, yet fundamental. The microbial microecosystem in the digestive tract is a perfect illustration.
The microbial digestive flora of an animal is in direct contact with the outside
environment. In fact, the intestinal tract can be thought of as a vast invagination of
the outside environment toward the interior of the animal. This interface allows
changes for both environments in the piglet. The consequences on the digestive
flora of the animal are then measurable (Midtvedt, 2002; Tournut, 1979;
Tannock, 1974). The imbalance of these ecosystems then defines the border
between normal and pathological.

Complexity and function of the digestive flora

The surface of the porcine intestine represents a gigantic platform for exchange
with the environment. The pig harbors around 1014 living microorganisms in its
digestive tract which is approximately 10 times more cells than found in the pig
itself.
The composition of the normal intestinal flora plays an important nutritional role
and can also influence the health of the animal. A microflora considered
normal and steady is support to the resistance of colonization by pathogens.
Some authors have termed this the shield effect (Nurmi, 1973). The microflora
also has some immunomodulation properties.
Moore and Holdeman were the first to quantify and characterize the complexity of
the digestive ecosystem (1974). From 20 fecal samples, they isolated 113 different
bacterial species. They also estimated that the bacterial population of the
digestive microflora constituted 400 to 500 different species and could
enumerate 1011 cfu/g (colony forming units/gram) of feces. Finegold et. al., found
similar results (1983).
They recovered dominant anaerobic flora including:
Bacteroides, Eubacterium, Clostridium, Ruminococcus, Peptostreptococcus,
Bifidobacterium, and Fusobacterium as well as the anaerobic-aerotolerant bacteria
of the Lactobacillus and Enterococcus genera. In addition they found facultative
anaerobic bacteria, mainly Escherichia coli.

Virus

environnment

Farm

Bacteroides

Bacteria

Yeast
Levures

Eubacterium

Bacillus

Sow

piglet

2.2 The digestive flora of the piglet

Peptococcus

Corynebacterium

Forme
incurves
Form incurved

room

Staphylococcus

Bifidobacterium

Streptococcus

house

Lactobacillus

Enterobacteries

meat

Veillonella

Spirochetes

Clostridium p
Fusobacterium

Figure 2 : Diagram of the rearing ecosystems affecting pork production.

Flore
fcale
exprim
Fecal
flora
in % e en%

Figure 3 : The fecal bacterial composition of the adult pig

(Gournier-Chteau, 1994)

12

* Colony Forming Units

13

The Porcine gastrointestinal ecosystem

The Porcine gastrointestinal ecosystem

The Porcine gastrointestinal ecosystem

Lactobacillus
E. coli
Streptococcus
Yeast

109
102
106
104

LIVER

JEJUNUM
pH 4 - 6

COLON
pH 6.5 7.5

pH 6.5 7
CECUM

STOMACH

Lactobacillus
E. coli
Streptococcus
Yeast

pH 5.5 6.5
ILEUM

107
104
104
104

+Lactobacillus
- E. coli

Streptococcus

- Bacteroides

Eubacterium

+Bifidobacterium
- Clostridium

Selenomonas
Yeast

106
106
107
108
1010
1010
1010
1010
104

Figure 4 : Presentation of some of the digestive organs of the pig and

their microbial species and populations

(Gournier-Chteau, 1994)

Figure 5 : Concentration and function of the intestinal pig flora and

its effects on the host (modified, according to Mitsuoka)

Figure 4 takes into account the distribution of the microorganisms and their known
effects on the animal : positive (+) or negative (-) :
Lactobacillus and Bibidobacterium primarily act as beneficial to improve
digestion, limit pathogenic microorganisms, and influence immunity.
Escherichia coli and Clostridium perfringens, Clostridium difficile can
have negative effects on the animal. They are capable of producing noxious
substances including: amines, indols, ammonia, phenols, and sulfurous anhydride.

14

Microorganisms can play an essential role in the host by reinforcing

protective effects or weakening these barriers.
Pathogenic microorganisms can cause sickness when a threshold is reached in the
host:
- Greater than 106 Clostridium perfringens per gram of intestinal content
- Greater than 105 Salmonella sp. per gram of intestinal content to trigger the
pathological process
One of the first functions of the intestinal microflora is to serve as a
protective barrier from the invasion of enteropathogenic bacteria in the
host (Raibaud and Ducluzeau, 1989; Chapoy, 1985).

15

The Porcine gastrointestinal ecosystem

3
Sequence of the Microbial Establishment
Sterile until birth, the digestive tract of the piglet is quickly colonized in the first 24
hours of life by the maternal microflora and environment (Tournut, 1993).
Three main phases of colonization:
Phase 1: 0-7 days:

The Imbalance

The aerobes and facultative anaerobes are replaced gradually by strict anaerobes.
After 24 hours, bacteria of the Escherichia sp. are found at 108 to 109 per gram of
intestinal contents (Smith, 1965). Lactobacilli appear in the first 4 to 5 days of
extra-uterine life (Raibaud and Ducluzeau, 1989; Smith, 1965).

- Changes in nutrition, stress and antibiotherapy can

strongly modify the composition of the intestinal flora.

Phase 2: 7-21 days:

- Antibiotics can be very effective but can also negatively

affect important commensal digestive flora

This phase is dominated by the proliferation of anaerobes as well as Clostridium sp.

and Bacteroides sp. Smaller populations of Eubacteria and Fusobacteria are found
along with variable populations of anaerobic Streptococcus.
Phase 3: 21-120 days:

- Growth promoting usage of antibiotics in the EU will be

banned January 1, 2006.
- There is a need to preserve this therapeutic arsenal while
developing other bacterial control methods.
- Probiotics can help to fight this imbalance.

This phase is characterized by the replacement of the gram positive anaerobes by

gram negative anaerobes. In the adult animal, Lactobacillus, Streptococcus,
Escherichia, Clostridium, and Bacteroides constitute the majority of the intestinal
flora.
After this sequence of flora establishment, the objective becomes a homeostatic state
in the gastrointestinal tract. Nevertheless, various disruptions can provoke an
imbalance in this homeostasis.

16

- Characteristics of probiotics :
safety
alive in the digestive tract
in a sufficient and efficacious dose
proven performance benefits
controllable

17

The Imbalnce

The Porcine gastrointestinal ecosystem

3. The Imbalance
Stress can also be considered as a potential source of microbial imbalance in the
pig. Work by Tournut et al., showed that when stress (constrained by corset) is
imposed on pigs of 20kg, the composition of the bacterial flora varies (1969).

3.1 Origin of the Imbalance

During rearing the balance of the intestinal microflora in pigs is continuously
subjected to disruptions. Different factors can all have an influence: nutritional
changes, rearing conditions, transportation, and the use of medications.
Changes in nutrition can strongly modify the composition of the intestinal flora.
Feed containing differing levels of carbohydrates and proteins can have different
effects on the digestive flora (Tzipori and Chandler, 1980). Likewise, feed
containing more dairy products and higher energy has a direct effect on the
microflora and on the animal. Studies have shown a decrease in diarrhea when
using these feedstuffs (Tzipori and Chandler, 1980). Other ingredients such as
soybean meal seem to show an increase in diarrhea. The absence of feed also seems
to play a role in these populations. Trials conducted by Tannock et. al., on the
intestinal flora of the mouse showed a variation in the microbial populations in the
digestive tract when feed was withheld (1974).

3,5

P < 0.05

3
2,5

P < 0.01

2
1,5
1
0,5
0

Localization

Observations

Stomach

lactobacilles

Microorganisms

Lactobacillus
NLF (a)(CD-1)

Jejunum-Ileum

coliforms

E. Coli (Ha/Icr)(b)

Sulfito-rducteurs
Sulfit reducer

Enterococcus
Entrocoques

Collibacillus
Colibacilles
Lot A Control

Lot B stressed

Figure 7 : Constraint by corset on pigs Imbalance of the flora

(Tournut, 1979)

Klebsiella (C57 BL)

Cecum

Colon

coliforms

No significant results

NLF (a)(CD-1)(b)
E. Coli (Ha/Icr)
Klebsiella (C57 BL)
-

Figure 6 : Modifications observed in the mouse gastrointestinal tract after a

Antibiotics, chemical substances produced by microorganisms, can inhibit

the growth of some microorganisms (bacteriostatic) and kill some
microorganisms (bactericidal). Antibiotics can be very effective but can also
negatively affect important commensal digestive flora. The selectivity
of antibiotics against pathogens is still somewhat of mystery (Chachaty and
Tancrde, 1999).

48-hour feed withdrawal

(Tannock et. al., 1974)
18

19

The Imbalnce

Log cfu/g

The Imbalnce

The Imbalnce

One of the first observable effects following administration of antibiotics is the

elimination of bacteria sensitive to the antibiotic. Other undesirable effects
can appear such as selection for resistant bacteria either from the preexisting flora
or by colonization from an outside source. The disappearance of the commensals
caused by the antibiotic can also be a problem because these commensals can serve
as gatekeepers of an efficient flora.
The situation then becomes extremely fragile as resistant bacteria and potential
pathogens take over the colonization. The antibiotic can have both an initial
positive action and a latter negative action that can provoke a lawlessness of the
digestive flora in the animal (Chachaty and Tancrde, 1999).
The rearing conditions can have an important impact on the digestive flora of
the piglet. Poor hygiene and environment become critical issues if handled
improperly (Madec, 1998).
The piglet is constantly submitted to various stressors that can imbalance its
digestive ecosystem. The fragility of the digestive microflora at birth represents a
critical period. The prevention of these imbalances is key to health and profitability.

3.2 Solutions

Origin of the
Imbalance

Consequences to the
piglet

Visible
Repercussions

Reference

STRESS OF BIRTH
AND WEANING

Imbalance of the
bacterial flora

Reduction in
performance

Tournut et al.,
1969

AGE AT WEANING

D17 : Villi height and

crypt depth
development reported
delayed at this age

Impact on the
absorptive
function of
nutrients by the
pig

Gu, 2002

ANTIBIOTICS

Action outside of
intended flora,
elimination of
commensals and
selection of
resistant bacteria

Impact on piglet
and potentially
human health

Bohnhoff, 1954
Chachaty and
Tancrde, 1999

DIET
COMPOSITION

Components that
affect the appearance
of diarrhea
(Soybeans, low
energy, dairy
products)

Decreased
performance

Tzipori and
Chandler, 1980

ENVIRONMENT
(T, AIR)

Temperature
fluctuations that
stress the pig

Decreased
performance

Madec, 1998

Antibiotics
Antibiotics have been used in animal production and by man since the turn of the
century. Since Flemings 1928 discovery of penicillin, antibiotics have been used for
many ailments.

Figure 8 : Examples of parameters that provoke an imbalance in the flora.

Growth promotion
Starting about 50 years ago, antibiotics have been used in subtherapeutic doses for
growth improvements in animal performance. This application raised some
concerns from European political figures in the late 1990s due to the concern of
antibiotic resistance.
These figures decided to withdraw the use of all subtherapeutic
antibiotics by January 1, 2006. However the use of therapeutic
antibiotics remains very important.

20

Therapeutic use
The use of therapeutic antibiotics remain very important. Some scientists argue that
a rationalization of therapeutic antibiotic usage must be considered in parallel to the
suppression of the antibiotic growth promoters. Antibiotics are efficient against
bacterial diseases and bacteria itself but might better serve as the ultimate control of
a disease situation. There is a need to preserve this therapeutic arsenal while
developing other bacterial control methods.

21

The Imbalnce

Probiotics
A reader who had some knowledge on this matter could be surprised,
according to my recommendations, to absorb big quantities of
microbes. All thought that the microbes were harmful. This belief is
erroneous. There are a lot of useful microbes. Elie Metchnikoff, 1907.
The science of probiotics began approximately 100 years ago with Russian biologist,
Elie Metchnikoff, the father of probiotics. New research and developments continue
to occur at an astounding rate. Scientists from all over the globe are expressing an
increasing interest in their evaluation.
The authors agree to define the probiotic concept as an administration of
living microbial cultures having an impact on the health of man or
animals and on their well-being while acting on the intestinal microbial
balance (Fuller, 1989).
For a microorganism to be considered a probiotic, some criteria must be met:
- It must remain viable in the digestive tract,
- It must be present in sufficient number (106-107cfu/g),
- It must not be pathogenic. The innocuity must be scientifically demonstrated,
- It must have statistically proven benefits,
- It must be easily analyzed.
A continuous daily ingestion is an efficient means to maintain an elevated probiotic
population in the digestive tract.

Saccharomyces cerevisiae boulardii

- Saccharomyces cerevisiae boulardii was discovered for the first
time in 1920 by Henri Boulard.
- The genus Saccharomyces has 4 differents
Saccharomyces
cerevisiae
has
thousands
of
Saccharomyces cerevisiae boulardii is unique.

species.
strains.

- Its intestinal action results in a real SHIELD EFFECT by :

neutralization of bacterial toxins of Clostridium difficile. This
bacteria is responsible for severe diarrhea. S.c. boulardii limits
toxin concentrations, increases local immunity and has a
protective effect on intestinal villi.
adherence of flagellate bacteria, due to the presence of
mannose receptors. These pathogens are eliminated by feces.
Moreover, lactic bacteria, a beneficial flora, are increased.
reinforcement of mucosal integrity and intestinal cells. S.c.
boulardii increases enterocyte enzymatic activity. This yeast has
a documented efficacy on villi height and crypt depth, enhancing
the assimilation of nutrients.

22

23

Saccharomyces cerevisiae boulardii

The Imbalnce

4. Saccharomyces cerevisiae boulardii

4.1 Origin of Saccharomyces cerevisiae boulardii
The S. c. boulardii story started around 1920, when a French microbiologist, H.
Boulard, made a surprising discovery in Indo-China. He noticed that consuming a
particular drink could alleviate signs of diarrhea in villagers afflicted by an epidemic
of cholera. This drink was made from tropical fruits such as lychee and mango.
Boulard isolated an active agent from this drink, which proved to be live yeast of
natural origin, and which is now known as S. c. boulardii.
Since then, many researchers world wide have reported the therapeutic properties
of this yeast, which has been found to be effective in both man and animals
(Figure 9).

50
40
30
20
10
0
19821984

19851987

19881990

19911993

19941996

19971999

20002002

Figure 9 : Evolution of the number of publications referring to S.

c. boulardii on Medline (National Library of Medicine).

24

4.2 Unique taxonomy and specificity

Many different types of Saccharomyces are used in the food and pharmaceutical
industry. Within the Saccharomyces genus, the different species must be
distinguished among the most frequently used. There are four distinct
species classified under Saccharomyces: S. bayanus, S. cerevisiae, S.
paradoxus, and S. pastorianus (Cardinali and Martini, 1994). Within S.
cerevisiae exist many different strains of which S. c. boulardii is one.
Mitochondrial DNA restriction assays (Malli et al., 2001) and microsatellite
typing techniques (Hennequin et al., 2001) confirm S. c. boulardii is a unique
strain of S. cerevisiae. The proper taxonomic name is therefore Saccharomyces
cerevisiae boulardii (Malli et al., 2001).

m : Marqueur de taille : Raoul (Appligene)

Saccharomyces
Ligne A
Lignes B G
Saccharomyces
Saccharomyces
Lignes H K
Saccharomyces
Lignes L R
Saccharomyces
Lignes S X
Saccharomyces
Ligne Y
Saccharomyces
Ligne Z

boulardii IVP 4271

cerevisiae IVP 4255 ; 4266 ; 4267 ; 4268 ; 4269 ; 4270
paradoxus IVP 4255 ; 4256 ; 4257 ; 4258
pastorianus IVP 4248 ; 4249 ; 4250 ; 4251 ; 4252 ; 4253 ; 4254
bayanus IVP 4259 ; 4260 ; 4261 ; 4262 ; 4263 ; 4264
boulardii IVP 4272
boulardii IVP 4273

Figure 10 : Electrophoresis gel of S. c. boulardii and the other species that

comprise the Saccharomyces genus digested with Hae III restriction

endonuclease (Malli et al., 2001)

Some taxonomic differences have been found associated specifically with

S. c. boulardii. The outer membrane of S. c. boulardii is richer in mannose, a
sugar permitting the binding of pathogenic bacteria that contain Type 1 fimbria,
than other yeasts of the same species (Gedek, 1999; Ofek, 1984). Numerous
publications have also identified the production of a specific 54-kilodalton
protease against the bacterial toxins produced by Clostridium difficile (Castagliuolo
et al., 1999, 1996).

25

Saccharomyces cerevisiae boulardii

National Library of Medicine, MEDLINE

Saccharomyces cerevisiae boulardii

Age of pigs involved:

1-7 days of age.

29% of the individual piglets were

toxin-positive.
C. difficile was the sole pathogen
detected in 19% of the piglets.

4.3 The SHIELD EFFECT of S. c. boulardii

year 2001

60
50
40
30
20

year 2001

2000 data (JVDI article) :

At least one toxin-positive pig was
detected in 55% of the cases

Iowa State University (VDL), 2001

Year 2000

Case:
group of young scouring pigs
from the same site

year 2000

Saccharomyces cerevisiae boulardii

Other researchers have found :

- a reduction of Clostridium perfringens (Awad, 2003),
- an increase in intestinal brush border membrane disaccharidases, an
increase in the synthesis of polyamines (Kollman, 2001), and positive effects on
intestinal immunity (Qamar et al., 2001; Rodrigues, 2000).

10
0

The intestinal flora of the pig provides a natural shield against invasion.
imbalances experienced by the pig serve to decrease its natural defenses.

The

Saccharomyces cerevisiae boulardii contributes to this shield effect by several

different mechanisms of action:
neutralization of bacterial toxins of Clostridium difficile,

Only pathogen
Pathogen associated

Figure 11 : Prevalence of Clostridium difficile in neonatal diarrhea

diagnostic for piglets (Yaeger and al., 2001)

adherence of flagellate bacteria,

reinforcement of the intestinal mucosa by metabolic, histological, and
immunological effects

Mode of action of Clostridium difficile :

NEUTRALIZATION of bacterial toxins of Clostridium difficile
Context :
Responsible for tetanus, botulism, or neonatal diarrhea in piglets, Clostridia are
potential pathogens for animals. Recent studies by Dr. Yaeger at Iowa State
University in the United States on C. difficile prevalence in piglets conclude that
these bacteria are extensively implied in cases of neonatal diarrhea. He recovered C.
difficile in 60% of the cases of neonatal diarrhea as a pathogenic partner with other
pathogens and in 40% of the cases it was the only pathogen recovered (Figure 11).

Clostridium difficile is a Gram positive strict anaerobe responsible for a type of

diarrhea called pseudo-membranous colitis in humans.
In vivo, its pathogenic action is mediated by the production of two toxins:
- A (enterotoxin),
- B (cytotoxin).
These toxins bind to specific glycoproteins in the intestinal mucous membrane.
This binding triggers the lesion process with disintegration of the actin filaments of
the enterocytes and increases the intracellular permeability.
Diarrhea is the most clinically observed sign. The final consequence is an
infiltration of the mucous membrane by polynuclear neutrophils and an edema of
the submucosal membrane.

26

27

Saccharomyces cerevisiae boulardii

Saccharomyces cerevisiae boulardii

Qamar et al., demonstrated that an administration of C. difficile toxin A to S. c.

boulardii treated mice gave an increase of immunoglobulins (IgA) by four
fold and an increase of IgM compared to non-treated mice (2001).
A trial on a swine farm that has a high prevalence of C. difficile showed a 30%
reduction in the toxin concentration with a reduction of clinical signs of
diarrhea in piglets after administration with S. c. boulardii (Delforge, 2003).
Figure 13 is a summary of different mechanisms of action of S. c. boulardii on C.
difficile.

Effect of C. difficile

C. difficile + S. c. boulardii
C. difficile non Toxinogenic clone

Figure 12 : Effects of C. difficile toxins on the intestinal villicytes.

(Castagliuolo, 1996)

Vitamin

Synthese

The pathogenicity of C. difficile expresses itself via its toxins. Saccharomyces

cerevisiae boulardii does not alter the population number but has been found to
reduce the amount of toxins produced (Castex et al., 1990).

A steric hindrance of mucosal toxin binding has also been elucidated.

In VEROS cells (kidney cells of African green monkeys), S. c. boulardii has been
shown to limit the implantation of C. difficile cells in the mucous membrane,
thereby preventing the liberation of toxins from the unattached cell (Tasteyre et al.,
2002).

S. c. boulardii

Toxinogenic C. difficile clones

Mode of action of S. c. boulardii on Clostridium difficile :

This mechanism of action involves the production of a specific protease by S. c.

boulardii that degrades toxins A and B of C. difficile (Castagliuolo et al., 1996, 1999).
This protease has been identified as a 54 kilodalton molecular weight serine
protease (Castagliuolo et al., 1999).
After administration of S. c. boulardii to C. difficile infected animals, the cecal
mucosa was more normal with a higher integrity of tight junctions (Castex, 1989,
1994).
In addition, S. c. boulardii exercised a protective effect on the villicytes of the
jejunum (Castex, 1989, 1994).

toxin A

protease

Digestion

toxin B

Synthese IgA

Digestion

4
Colonic
mucosa

1
+

_
Tox A
Tox B

_
_

IL 8

Chemoattractant

neutrophils

Synthesi
s

Tight junctions (ZO-1 protein)

Eicosanodes

1 - 54kDa protease which digests toxins A and B.

2 - The reinforcement of the intestinal epithelial barrier through the tight-junction stabilization
(ZO-1 protein)
3 - The induction of non-toxinogenic C. difficile clones outcome, which modulate the toxin
synthesis pathway of toxinogenic strains
4 - The enhancing of the mucosal immune response by the stimulation of specific anti-toxin A IgA
synthesis which inhibit the adhesion of harmful bacteria and toxins
5 - The direct action on the toxin synthesis pathway through the release of a vitamin.

Figure 13 : Schematic illustrations of different mechanisms of S. boulardii

action for the control of C. difficile associated diarrhea

28

29

Saccharomyces cerevisiae boulardii

4
flagellate bacteria

The binding sites at the surface of S. c. boulardii have been studied in vitro using
histological methods (Gedek, 1999).
Using electron microscopy, it has been shown that the lectin sites of Type 1
fimbria of E. coli bind more to the surface of S.c. boulardii than other
yeast strains of the same species. These lectins bind the mannose sugars
present on the yeast as shown in Figure 14 (Gedek, 1999).

Enzymatic Activity :
Brush border membrane disaccharidases hydrolyze disaccharides to
monosaccharides. A lack of these disaccharidases can lead to a malabsorption of
sugars provoking :
- An influx of water into the lumen of the intestine, responsible for the liquid
character of diarrhea,
- A proliferation of fermentative microflora with a conversion of sugars into
organic acids.
- Saccharomyces cerevisiae boulardii has been shown to increase the
activity of lactase, sucrase, and maltase at the intestinal level (Buts et al.,
1986).

E. coli

Activity
350
moles/
min/g protein 300
treatments

250
yeast

200

Before
After

150
Figure 14 : Adherence of E. coli 0157:H7 to the

surface of S. c. boulardii
(Gedek, 1999)

100
50
0

Several in vivo studies measured the impact of S.c. boulardii on colonization by

Salmonella in broilers (Awaad, 2003 ; Line, 1997). The results show frequency of
colonization by Salmonella reduced 65 % with S.c. boulardii compared to
control. An agglutination of Salmonella to S.c. boulardii might explain this
reduction, as certain Salmonella are known to contain Type 1 fimbriae that bind to
the mannose receptors present in the cell wall of S. c. boulardii (Mirelman, 1980).
S.c. boulardii limits intestinal attachment of these pathogens and
consequently limits bacterial colonization.

30

Lactase

Sucrase

Maltase

Figure 15 : Evolution of the activity of lactase, sucrase, and

maltase at intestinal levels after 14 days of oral ingestion of
S. c. boulardii
(Buts et al., 1986)

31

Saccharomyces cerevisiae boulardii

ADHERENCE of

CNRI

Saccharomyces cerevisiae boulardii

REINFORCEMENT of mucosal integrity and epithelial cells

4
The polyamines spermine and spermidine are biologically active molecules
implicated in cell proliferation. It has been shown that S. c. boulardii increased
levels of spermine and spermidine (Buts et al., 1994). This increase is linked to the
beneficial effects on the mucous membrane, notably an increase in villous height
and crypt depth.

260

8
7

220
200

160
140

120

100

Crypt depth

2
Coliforms

on intestinal microbial populations

in the digestive tract.

110

Villi height

Lactobacilli

* p<0.01

Figure 17 : Effect of S.c. boulardii

120

(m) 180

130

240

(Biocentrum Agrivet, 2001)

Figure 18 : Effect of S. c. boulardii

on epithelial cells in intestinal tract

(P<0.01)
(Vet University of Milan, 2002)

100
90
80
Spermidine
Spermine
Figure 16 : Effect of S. c. boulardii on polyamine concentrations in

the jejunal mucosa. Both polyamines were significantly increased

(P<0.05) (Buts et al., 1994).

Effect on microbial populations

While S. c. boulardii limits intestinal pathogenic flora, it also increases
concentrations of beneficial flora, most notably bactobacilli (Figure 17). It
contributes to a better balance of digestive flora.

Ileal villi with

Levucell SB

Ileal villi in Control group

Maturation of epithelial cells

A trial conducted at Milan University (Figure 18) shows that Saccharomyces
cerevisiae boulardii significantly increases crypt depth and villi height in pigs
(Figure 19).

Figure 19 : Electron micrographs of intestinal villi

As a consequence, intestinal absorption is improved as is feed efficiency, thus

optimizing animal growth.

32

33

Saccharomyces cerevisiae boulardii

Saccharomyces cerevisiae boulardii

5
m

**

400
Control
Levucell SB

Conclusion
*

300

*
*

200

Consumers are becoming more aware of animal production and it implications in

foodborne diseases.

100
* p < 0.05

Producers have the objectives of:

** p < 0.01

Villi

Crypts

Villi

Crypts

Biocentrum
Agrivet
Biocentre
Belgium (2001)
B

Vet UtyMilan
of Milano

Animals

10

20

Production
performance in
piglets
(% / control)

ADG : + 15%
FCR : + 6%

DWG : + 6.5%

Italy (2002)

Figure 20 : Correlation between the effect on epithelial

cells and production performance in piglets

(University of Milano and Biocentrum Agrivet)

Production performance :

- Becoming more competitive,

- maintaining public health associated with animal production. One solution is
to master the sanitary conditions in the production environment while maintaining
performance.
Saccharomyces cerevisiae boulardii links economic performance with food safety.
This yeast has a mode of action referenced in more than 145 international
publications :
- With the protease secreted, it can neutralize bacterial toxins of Clostridium
difficile, bacteria reponsible for diarrhea and colitis associated with antibiotic
usage;
- S. c boulardii can expulse in feces flagellate and pathogenic bacteria
bound to its wall ;
- S. c. boulardii reinforces mucus production and integrity of intestinal
cell walls by stimulation of enzymatic activities, an increase in local immune
response, an increase in maturity of epithelial cells This contributes to a better
utilization of the feed (villi height increased).
Finally, the economic objectives (growth, health, feed efficiency) coincide with food
safety objectives to provide a positive image associated with decreased antibiotic
usage. Saccharomyces cerevisiae boulardii is a hygienic tool at the farm
level.

Two trials, summarized in Figure 20, show the relation between the effects on
epithelials cells and production performance.
Average daily gain in piglets supplemented with S. c. boulardii increased 6.5 to
15%, while feed conversion was 6% more efficient.
As a consequence, Saccharomyces cerevisiae boulardii offers measurable
results on piglet growth.

34

35

Conclusion

Saccharomyces cerevisiae boulardii

6
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