Strain deposit: I-1722 - Collection Nationale de

Cultures de Microorganismes CNCM, Institut

Pasteur, France.
Cell morphology: Rod; non-sporulating; nonmotile; tending to form pairs or short chains;
GRAM-positive, homofermentative, facultative
anaerobe. In laboratory conditions, grows well in
commercially available media for lactic acid
bacteria (MRS, Mann Rogosa and Sharp) at 37 C
(98 F) and forms white colonies.

Phenotypic Identification

General Information
The Lactobacillus genus currently consists of over
125 species and encompasses a wide variety of
organisms. These organisms are strictly
fermentative, aerotolerant or anaerobic and have
complex nutritional requirements. Using glucose
as a carbon source, lactobacilli may be either
homofermentative (producing more than 85% of
fermentative products as lactic acid) or
heterofermentative (producing lactic acid, carbon
dioxide, ethanol and/or acetic acid).

General Properties of Lactobacilli

Maintain a healthy and balanced intestinal
microbiota and reduce the incidence of intestinal
infections.
Frequently recommended to prevent
unbalanced intestinal microflora and antibioticassociated diarrhea.
Contribute to the modulation of specific and
non specific immune responses.

Carbohydrate fermentation pattern: API 50 CHL

Biochemical characterization: API Zym

Genotypic Identification
Strain Rosell-52 was formerly identified as
Lactobacillus acidophilus and reclassified as
Lactobacillus helveticus in 2006 following DNADNA hybridization. This reclassification does not
imply any difference in the intrinsic properties of
the strain and does not contradict any of the data
obtained with Lactobacillus Rosell-52 with respect
to safety and efficacy. Multi-locus sequence typing
of Lactobacillus Rosell-52 confirms it is a unique
strain of Lactobacillus helveticus.
Furthermore, Lactobacillus Rosell-52 is still very
closely related genotypically and phenotypically to
the Lactobacillus acidophilus group A complex [5].
16S rDNA sequence and DNA-DNA hybridization
confirm the species designation (Lactobacillus
helveticus) of Rosell-52 (Fig. 1).

Lactobacillus Rosell-52
Lactobacillus helveticus is a homofermentative
thermophilic lactic acid bacterium that is widely
used in the manufacture of fermented dairy
product including cooked cheeses. [1].

Strain identification:
Lactobacillus helveticus R0052 (Rosell-52)
Origin: Dairy
Isolation date: 1990
Lactic acid produced: DL
Other molecules produced: Acetate, folate.
Lactobacillus helveticus is known to possess
elevated amounts of proteolytic activity [2,3,4],
which maximizes the probabilities of releasing
bioactive peptide.

Figure 1 Rosell-52 Phylogenetic tree based on 16S rDNA

sequences.

In addition, the complete Lactobacillus Rosell-52

genome has been sequenced and annotated (Fig.
2).

been performed with products containing Rosell52 and no adverse effects have been observed.
[9]
Antibiotic resistance:
MIC (Minimum Inhibitory Concentration) has
been completed using broth microdilution as per
the recommended methods [10]. Microbiological
breakpoints were suggested by the FEEDAP Panel
(EFSA) for Lactobacillus helveticus in their
Technical guidance - Update of the criteria used
in the assessment of bacterial resistance to
antibiotics of human or veterinary importance Prepared by the Panel on Additives and Products
or Substances Used in Animal Feed, adopted on
June 18, 2008 [11]. Furthermore, using
microarray screening for 170 known antibiotic
resistance genes, Institut Rosell has proven that
Lactobacillus Rosell-52 does not contain any
known antibiotic resistance genes (Fig. 3).

Figure 2 Rosell-52 Genome Atlas. Courtesy of Dr. Eric

Altermann, Palmerston North, New Zealand.

Strain Safety
All our plants comply with Good Manufacturing
Practice requirements.
A public consultation on QPS (Qualified
Presumption of Safety) was published by the EFSA
(European Food Safety Authority) with a list of
species proposed for QPS granting. Lactobacillus
helveticus is among the strains proposed by the
Scientific Committee for QPS status [6]. The
International Dairy Federation (IDF), in
collaboration with the European Food and Feed
Cultures Association (EFFCA), assembled a list of
microorganisms with a documented history of
safe use in food [7]. Lactobacillus helveticus is
listed on this inventory. In addition, Institut Rosell
maintains safety files (Active Substance Master
File) on Lactobacillus Rosell-52 in CTD format
(Common Technical Document, Module 4), which
are deposited with government health agencies
and regularly updated in accordance with the
results of ongoing research. There have been no
reported adverse reactions or side effects with
the use of Lactobacillus Rosell-52.
Toxicological studies:
In vivo rat studies have been performed involving
28-day feeding studies (high dosage: 1 to 2 billion
CFU/day for 28 days by gavage; 2 billion CFU/day
per 200 g rat represents about 1,400 billion
CFU/day per 70 kg person). These studies have
been performed on Rosell-52 [8] and no
toxicological symptoms or abnormalities were
observed. Human safety and kinetics trials have

Figure 3 Proposed scheme for the antimicrobial resistance

assessment of a bacterial strain (FEEDAP 18/06/2008) [11].

Technical Properties
Lactobacillus helveticus is a fastidious organism
requiring exogenous supplies of specific carbon
and nitrogen sources, nucleotides, vitamins, and
minerals for growth [12,13]. Our team of
fermentation specialists optimizes growth
parameters, cryo-protection conditions and
environmental controls to obtain highly
concentrated, viable, freeze-dried stable cell
cultures.
Lactobacillus Rosell-52 is available in different
forms. Please, do not hesitate to contact our sales
representatives for more information.

Gastrointestinal Survival
Microorganisms chosen to be incorporated into
probiotic preparations should remain alive until
they reach their target site of action - the lower
small intestine. In order to do so, they must pass
through the stomach and the upper gastrointestinal
tract alive.
Resistance to gastric acidity and bile: Evaluation of
their resistance to stomach acidity and biliary salts
is one of the fundamental criteria for probiotic
selection. After 120 minutes in acidic conditions:
53% survival rate (pH 4); 47% survival rate (pH 3).
Lactobacillus Rosell-52 is able to survive and grow
in high concentrations of bile and, in vivo, should
reach the distal end of the small intestine without
damage [14].
A dynamic model of the human upper
gastrointestinal (GI) tract, the in vitro Digestive
System (IViDiS) model, was designed to better
simulate conditions of ingestion and digestion, by
including a food matrix as part of the model design.
The dynamic model consists of two reactors
maintained at 37 C, one simulating stomach
conditions and the other simulating duodenum
conditions [14].

Figure 4 Adhesion of Rosell-52 to T84 epithelial cells. Courtesy

of Dr. Phil Sherman, Hospital for Sick Children, Toronto,
Canada.

Survival in faecal samples after transit:

Lactobacillus Rosell-52 provided as a dietary
supplement can survive passage through the human
intestinal tract and no alteration is observed in
microbiota of healthy subjects [9].

Efficacy Studies
There are basically three mechanisms important to
consider:
The stimulation of the epithelial cell proliferation
and mucin production which enhance the barrier to
translocation of infectious agents.
The inhibition of pathogenic bacteria growth and
adhesion.
The production of specific and non-specific
immune responses and the alleviation of
inflammation.

The First Line of Defense

Survival of Rosell-52 was as expected for a 90

minute transit time. [1] Rosell-52 remains stable
under normal stomach and duodenal conditions.
Rosell-52 can be protected thanks our two patented
technologies: STAR and Probiocap.
Adhesion capacity: Lactobacillus Rosell-52 is able to
bind strongly to intestinal epithelial cell lines (T84)
which may be important for competition with
pathogens, stimulation of mucous production and
the modulation of the host immune system (Fig 4)
[15,16].

Lactobacillus Rosell-52 maintains the intestinal

barrier (control of the translocation).
Escherichia coli causes acute diarrhea, hemorrhagic
colitis and hemolytic uremic syndrome. In vitro tests
show that Lactobacillus Rosell-52 is able to inhibit
the adhesion to intestinal epithelial cells (T84) of
both enteropathogenic E. coli O127:H6 and
enterohaemorrhagic E. coli O157:H7 in a
concentration-dependent manner.
The adhesion of E. coli to epithelial cells causes a
cytoskeletal rearrangement of the epithelial cells,
which in turn induces them to pull apart and
compromises the tight junctions. When the
intestinal barrier is compromised, infectious agents
can translocate the barrier and leaky gut-like
symptoms could arise [16].
In challenge tests with pathogenic bacteria,
Lactobacillus Rosell-52 has been shown to protect
the integrity of the epithelium by limiting the loss of
trans-epithelial electrical resistance (TEER), a
measure of barrier integrity [16].

Lactobacillus Rosell-52 and the Pathogenic

Bacteria

Beneficial Modulation of the Immune

System

Lactobacillus Rosell-52 inhibits intestinal

pathogens.
Citrobacter rodentium is a member of the
Enterobacteriaceae and is a close relative of
Escherichia coli. It is a host-restricted mouse
pathogen and is used as a model system to study
the pathogenic mechanisms of enteropathogenic
and enterohaemorrhagic E. coli. During in vitro
study, Lactobacillus Rosell-52 incubated with
Citrobacter rodentium inhibited its growth in a
timedependent manner. This inhibition was
observed in the presence of probiotics plus
culture supernatants or that of culture
supernatants alone. The pH of co-culture medium
(pH 6.5) did not differ from that of Citrobacter
rodentium alone (pH 6.5), which suggests that
growth inhibitory effects could be mediated
through a bacterial product secreted into the
culture supernatant [17].

The intestine is one of the most important

lymphoid organs in the body. Its surface area
represents about 300 m and it contains 70% of
the immune cells of the organism. The primary
site of action for probiotics is the intestinal wall,
where they interact with epithelial and dendritic
cells and modulate the production of cytokines by
the underlying immune cells.
The intestinal immune system has oral tolerance
functions to suppress immune responses against
foreign proteins (of bacterial or food origin) that
are commonly found in the digestive tract. This
prevents over-sensitivity to food and minimizes
chronic digestive tract inflammation, such as
occurs in Ulcerative Colitis. On the other hand, the
intestinal immune system must promote
protective immune responses against pathogenic
microorganisms. Antibody synthesis is a key
component of protective immunity in the gut.

Lactobacillus Rosell-52 competes with pathogens

for adhesion to epithelial cells.
Surface-layer proteins (Slps) are located in a
paracrystalline layer outside the bacterial cell
wall. Lactobacillus Rosell-52 has a strong adhesion
capacity on intestinal epithelial cells due to its
specific surface proteins called S-proteins [18].
This strong adhesion capacity allows Rosell-52 to
occupy the attachment sites of pathogenic
bacteria like Enteropathogenic E. coli (EPEC) and
Enterohemorrhagic Escherichia coli (EHEC) on
intestinal epithelial cells, thereby avoiding
pathogen adhesion and translocation [18].

Lactobacillus Rosell-52 increases circulating

antibodies IgM & IgG.
IgM is the first antibody produced when a body is
challenged by antigens. IgG antibodies are
predominantly involved in the secondary antibody
response (the main antibody involved in primary
response is IgM), which occurs approximately one
month following antigen recognition. Thus the
presence of specific IgG generally corresponds to
maturation of the antibody response. Studies
have shown that Rosell-52 is able to induce the
production of non-specific polyclonal antibodies
(IgM and IgG) by murine splenocytes, in vitro,
therefore increasing the pool of antibodies able to
react quickly to infection [22].

Helicobacter pylori is a gastrointestinal pathogen

that can cause gastritis, peptic ulcers and is linked
to gastric adenocarcinoma. One study showed
that Lactobacillus Rosell-52 was also able to
partially inhibit adherence of H. pylori to
squamous oesophageal epithelial cells in a dosedependant manner. This inhibition was similar
under conditions of exclusion (R0052 is preincubated) and competition (R0052 and H. pylori
are coincubated). Lactobacillus Rosell-52 was
unable to displace H. pylori after it had adhered to
the cells [19]. Moreover, there is a rising
resistance of H. pylori strains to antibiotic what it
places the patient at higher risk for complications
such as post-antibiotic infections. The addition of
Rosell-52 to eradication therapy improves
eradication efficacy to 94,3% and reduces
complications after antibiotic therapy [20,21].

Lactobacillus
Rosell-52
modulates
the
production of cytokines by the human infected
intestinal epithelial cells.
The production of the pro-inflammatory cytokine
IL-8 by the HT-29 cell line (gastric epithelial cells
stimulated
by
pathogen
presence
or
inflammation), whose function is to stimulate
trans-endothelial migration of neutrophils to a
site of infection, is significantly down-regulated by
Lactobacillus Rosell-52 [23]. But Lactobacillus
Rosell-52 also prevents overexpression of RANTES
by gastric epithelial cells stimulated by pathogen
presence or inflammation. Lactobacillus Rosell-52
has no direct effect on TNF- and an insignificant
effect on the down-regulation of TNF-
(transforming growth factor) production by HT-29
cells lines when challenged with E.coli. [23]

Enterohemorrhagic Escherichia coli (EHEC) disease

pathogenesis includes subversion of epithelial cell
signal transduction cascades involved in host innate
immune responses to infection (STAT-1) in multiple
cell lines. For instance, EHEC inhibits interferon (IFN)-stimulated tyrosine phosphorylation of signal
transducer and activator of transcription-1 Gene
knockout mice provide evidence that the IFN-
Jak1,2STAT-1 signal transduction cascade is an
essential innate immune response required to
combat microbial infections [24].
It was shown that Lactobacillus Rosell-52 maintains
IFN-Jak1,2STAT-1 activation following EHEC O157
:H7 infection of multiple epithelial cell lines. Viable
Lactobacillus Rosell-52 is required for protection,
while contact with epithelial cells is not necessary.
Future research efforts should focus on
characterizing the molecular interactions between
this probiotic and host epithelial cells [24].
Lactobacillus Rosell-52 decreases a parasite
(nematode) Th2 & Th1-type induced response.
Four days after the Nippostrongylus brasiliensis
parasite infection in rats, a strong increase in both
Th-2 (IL-4, IL-5, IL-6, IL-10) and Th-1 (TNF-, IFN-)
cytokine levels is observed in the mid jejunum
mucosa. This is probably linked to the early postinfective stage recovery. Giving Rosell-52 treatment
to the infected animals significantly decreases the N.
brasiliensis effect on intestinal mucosa cytokine
levels and the values obtained are similar to
uninfected controls [24]. Overall, the results from
the N. brasiliensis infection study suggest that the
probiotic treatment exerts an immunomodulatory
effect which can positively influence functional
parameters in either Th2 or Th1 oriented
physiopathological conditions. [23,25,26,27]

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