TM 51-1e

Lactobacillus rhamnosus Lr-32

CHARACTERISTICS OF THE bacteria, and several published studies trations of bile salts found in the small
GENUS have addressed their safety (3-7). intestine.
Lactobacillus rhamnosus has existed in More specifically, L. rhamnosus is listed In vitro studies have shown that
cheese for hundreds of years and is also in the Inventory of Microorganisms With L. rhamnosus Lr-32 is resistant to low pH
one of the most common Lactobacillus Documented History of Use in Human conditions and survive the presence of
species in breast-fed infants (1). T
  he strain Food (8) and the Qualified Presumption bile at concentrations present in the duo-
comprises Gram-positive, facultatively of Safety list of the European Food Safety denum.
anaerobic, non-motile and non-spore- Authority (9).
forming, rod-shaped microorganisms. In addition to a long history of safe Adhesion to intestinal mucosa
human consumption, no acquired Interaction with the intestinal mucosa
antibiotic resistance was detected in is considered important for a number
L. rhamnosus Lr-32 during screening by of reasons. Binding to the intestinal
the EU-funded PROSAFE project. mucosa may prolong the time a probiotic
strain can reside in the intestine. T  his
GASTROINTESTINAL interaction with the mucosa brings
PERFORMANCE the probiotic in close contact with the
Resistance to acid and bile intestinal immune system, giving it a bet-
According to the generally accepted ter opportunity to modulate the immune
definition of a probiotic, the probiotic response. It may also protect against
SELECTION AND TAXONOMY microorganism should be viable at the enteric pathogens by limiting their ability
L. rhamnosus was originally thought to be time of ingestion to confer a health to colonize the intestine.
a sub-species of L. casei. Later genetic re- benefit.  Although not explicitly stated, this Currently, adherence is measured using
search found it to be a species in its own definition implies that a probiotic should two in vitro cell lines, Caco-2 and HT-29.
right.  As a result, in 1989 its taxonomic survive GI tract passage and, according to While this is not a thorough test of the
name was changed from L. casei subsp. some, colonize the host epithelium. ability of probiotics to adhere to intestinal
rhamnosus to L. rhamnosus (2). A variety of traits are believed to be mucosa in the body, attachment to these
L. rhamnosus Lr-32 has been genetically relevant for surviving GI tract passage, cell lines is considered a good indicator of
characterised and properly classified as the most important of which is tolerance their potential to attach.
L. rhamnosus by independent labs using both to the highly acidic conditions L. rhamnosus Lr-32 demonstrated
modern genotypic methods including present in the stomach and to concen- excellent adhesion to human epithelial
16S rRNA gene sequence analysis. cell lines applied in in vitro studies.
Originally isolated from an unknown
source, the strain has been deposited in
the American Type Culture Collection as +++
Acid tolerance (>70% survival in hydrochloric acid and pepsin (1%) at pH 3 for 1h at 37ºC)
SD5217.
++++
Bile salt tolerance (>80% survival in 0.3% bile salt containing medium)
SAFE FOR CONSUMPTION
+++
Lactic acid bacteria have long been Pepsin resistance (>40% in 0.3% pepsin containing medium at pH 2 for 1h)
considered safe and suitable for human +++
Pancreatin resistance
consumption.Very few instances of (>60% survival in 0.1% pancreatin containing medium at pH 8 for 2h)
infection have been associated with these Selected characteristics of L. rhamnosus Lr-32 (internally generated data):
++++ Excellent; +++ Very good; ++ Good; + Fair
 Adherence to HT-29: ++++ L/D- lactic acid production Modulation of the immune system is
human intestinal
cells in vitro Caco-2: ++++ Lactic acid is the most important meta- an area of intense study in relation to the
Selected characteristics of L. rhamnosus Lr-32 bolic end product of fermentation proc- Danisco probiotic range. T  he goal is to
(internally generated data): ++++ Excellent; esses by lactic acid bacteria and other understand how each strain contributes
+++ Very good; ++ Good; + Fair
microorganisms. For thousands of years, to the maintenance and balance of opti-
lactic acid fermentation has been used in mal immune function. T   he immune sys-
Inhibition of pathogens the production of fermented foods. tem is controlled by compounds known
The protective role of probiotic bacteria Due to its molecular structure, lactic as cytokines. Cytokines are hormone-like
against gastrointestinal pathogens is one acid has two optical isomers. One is proteins made by cells that affect the
of the most important properties for known as L(+)-lactic acid and the other, behaviour of other cells and, thereby, play
therapeutic modulation of the enteric its mirror image, is D(-)-lactic acid. an important role in the regulation of
microbiota. Probiotics are able to inhibit, In humans, animals, plants, and micro- immune system functions.
displace and to compete with pathogens, organisms, L(+)-lactic acid is a normal
but the abilities are strain dependent. intermediate or end product of the car- In vitro studies
The putative mechanisms of action bohydrate and amino acid metabolisms. It In vitro assays are widely used to define
of probiotic strains against pathogenic is important for the generation of energy the cytokine expression profiles of
microorganisms include the production under anaerobic conditions. probiotics and, thereby determine their
of inhibitory compounds, competition In the organs of humans and animals, immunological effects. By measuring
with the pathogen for adhesion sites, the endogenous synthesis of D(-)-lactic the impact of probiotic bacteria during
or nutritional sources, inhibition of the acid is very low in quantity. T
  he isomer interaction with cytokine-expressing
production or action of bacterial toxins, is normally present in the blood of peripheral blood mononucleocytes
ability to coaggregate with pathogens and mammals at nanomolar concentrations (PBMCs), information is generated that is
stimulation of immunoglobulin A. and may be formed from methylglyoxal, useful in determining the ability of each
In vitro inhibition is usually investigated derived from lipid or amino acid metabo- strain to contribute to balanced immune
using an agar inhibition assay, where soft lism. health.
agar containing the pathogen is laid over L. rhamnosus only produces L(+)-lactic L. rhamnosus Lr-32 was investigated in
colonies of probiotic cultures, and inhibi- acid. vitro for its ability to induce the PBMC
tion expressed as the zones of inhibition secretion of selected cytokines: inter-
developing around the colonies. L/D-lactic acid 100/0 leukin IL-10 and IL-12. T   he results were
This effect may be due to the produc- production Boehringer Mannheim/ compared with Lactococcus lactis, a
tion of acids, hydrogen peroxide, bacte- R-Biopharm D-lactic acid/ starter culture commonly used in the
Molar ratio L-lactic acid UV-method
riocins and other substances that act as production of various fermented foods,
Internally generated data
antibiotic agents as well as competition and Escherichia coli, a common member
for nutrients. It should be pointed out of the intestinal microbiota. IL-10 plays a
that extending such results to the in vivo key role in the control of inflammatory
situation is not straightforward. IMMUNOMODULATION responses to intestinal antigens.
The assessment in below table is based An immune system that functions opti- L. rhamnosus Lr-32 was found to induce
on an in vitro assay. mally is an important safeguard against in- IL-10 to a significantly higher degree than
L. rhamnosus Lr-32 displayed in vitro fectious and non-infectious diseases. T   he Lc. lactis and to a higher degree than E.
inhibition of selected pathogens. intestinal microbiota represent one of coli. IL-12 was induced to a lower degree
the key elements in the body’s immune than Lc. lactis, but higher then E. coli
Salmonella typhimurium: ++ defence system. (figure 1). T  his indicates that L. rhamnosus
Pathogen Staphylococcus aureus: ++++ Probiotic bacteria with the ability to Lr-32 has anti-inflammatory properties
inhibition
in vitro Escherichia coli: +++ modulate certain immune functions may (10).
Listeria monocytogenes: ++ improve the response to oral vaccination,
Selected characteristics of L. rhamnosus Lr-32 shorten the duration or reduce the risk Animal studies
(internally generated data): ++++ Excellent; of certain types of infection, or reduce In line with the results above,
+++ Very good; ++ Good; + Fair
the risk of, or alleviate the symptoms of, L. rhamnosus Lr-32 has further dem-
allergy and other immune-based condi- onstrated an ability to modulate the
tions. immune system in an inflammation
animal model, validating its ability to

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 IL-10 IL-12 absence of the specific antibiotic target or
2500 1000 is a consequence of natural selection.
900
Antibiotic resistance can be defined
2000 800
700
as the ability of some bacteria to survive
1500 600 or even grow in the presence of certain
pg/ml

pg/ml
500 substances that usually inhibit or kill other
1000 400
bacteria. T  his resistance may be:
300
500 200 Inherent or intrinsic: most, if not all,
100 strains of a certain bacterial species are
0 0 not normally susceptible to a certain
Lr-32 Lc. lactis E. coli Lr-32 Lc. lactis E. coli
antibiotic. T  he antibiotic has no effect on
Figure 1. In vitro cytokine expression of L. rhamnosus Lr-32 (10). these cells, being unable to kill or inhibit
the bacterium.
Acquired: most strains of a bacterial
contribute to a balanced immune system. untreated DCs did not rescue mice from species are usually susceptible to a given
Figure 2 demonstrates the degree of colitis, intra-peritoneal administration of antibiotic. However some strains may
protection from a chemically-induced DCs treated with L. rhamnosus Lr-32 led be resistant, having adapted to survive
intestinal inflammation. L. rhamnosus Lr-32 to a considerable reduction in the colitis, antibiotic exposure. Possible explanations
has led to a considerable reduction in with reduced weight loss, improved clini- for this include:
colitis symptoms and exerts significant cal parameters and a significant reduction • A mutation in the gene coding for the
protection from intestinal inflammation, in macroscopic inflammation scores antibiotic’s target can make an antibi-
demonstrating its ability to interact with (figure 3) (11). otic less efficient. T
  his type of antibiotic
and balance the intestinal mucosal im- resistance is usually not transferable.
mune response (10). ANTIBIOTIC RESISTANCE • A resistance gene may have been
L. rhamnosus Lr-32 was further included PATTERNS acquired from a bacterium.
in a study to investigate the role of den- Antibiotic susceptibility patterns are an Of the acquired resistances, the latter is
tritic cells (DCs) in the anti-inflammatory important means of demonstrating the of most concern, as it may also be passed
potential of probiotic bacteria. DCs be- potential of an organism to be readily on to other (potentially pathogenic)
long to the group of antigen-presenting inactivated by the antibiotics used in bacteria.
cells (APC) that play a central role in human therapy. Much concern has arisen in recent
orchestrating immune responses to own Antibiotic resistance is a natural prop- years regarding vancomycin resistance,
and foreign antigens. It has been shown erty of microorganisms and existed be- as vancomycin-resistant enterococci
that, after activation with different stimuli, fore antibiotics became used by humans. are a leading cause of hospital-acquired
DCs achieve maturation, leading to func- In many cases, resistance is due to the infections and are refractory to treat-
tional and phenotypic changes.
In this study it was demonstrated
that probiotic-treated DCs conferred
**
protection against TNBS-induced colitis
in mice.  While the administration of NS **
8
Wallace score

6
Colitis protection
70 4
60
50 2
% protection

40
30 0
20 Treatment None DC Lr-32 DC
10
0 Protection, % 5 74
-10
-20 Figure 3. Protective effect of intra-peritoneal administration of LAB-treated DCs on acute TNBS-induced
Lr-32 Lc. lactis E. coli
colitis in BALB/c mice.  Wallace inflammation scores were calculated after a TNBS challenge in mice
Figure 2. Percentage of protection in an acute either not treated (none) or intra-peritoneally injected with untreated DCs (DC) or DCs treated with L.
murine model of inflammation (TNBS) (9). rhamnosus Lr-32 (Lr-32 DC) (11).

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ment. T  he transmissible nature of genetic The antibiotic susceptibility patterns 5. Salminen, S., von Wright, A., Morelli,
elements that encode vancomycin resist- for L. rhamnosus Lr-32 are summarised in L., Marteau, P., Brassart, D., de Vos,
ance in these enterococci is an important table 1. W.M., Fonden, R., Saxelin, M., Collins, K.,
mechanism of pathogenicity. Mogensen, G., Birkeland, S.-E. & Mattila-
Resistance to vancomycin in certain BENEFIT SUMMARY Sandholm,T. (1998). Demonstration of
lactobacilli, including L. rhamnosus, Based on the data supporting the safety of probiotics-a review. Int. J. Food
pediococci and leuconostoc is due to qualities of the L. rhamnosus Lr-32 strain, Prot. 44:93-106.
intrinsic factors related to the composi- the health-related attributes can be sum- 6. Borriello, S.P., Hammes, W.P., Holzapfel,
tion of their cell wall, and not due to any marised as follows: W., Marteau, P., Schrezenmeir, J.,Vaara, M.
transmissible elements (12). L. rhamnosus • Well suited to intestinal survival & Valtonen,V. (2003). Safety of probiotics
Lr-32 has been confirmed through PCR -- High tolerance of acid and intestinal that contain lactobacilli or bifidobacteria.
testing to be free of Enterococcus-like bile Clin. Infect. Dis. 36:775-780.
vancomycin-resistance genes. -- Strong adhesion to intestinal cell 7. Gueimonde, M., Ouwehand, A. C. &
As yet no case of antibiotic resistance lines Salminen, S. (2004). Safety of probiot-
transfer has ever been identified and • Beneficial modulation of immune func- ics. Scandinavian Journal of Nutrition.
reported for lactic acid bacteria used in tions 48:42-48.
foods and feed. -- L. rhamnosus Lr-32 may influence 8. Mogensen, G., Salminen, S., O’Brien, J.,
immune regulation, as demonstrated Ouwehand, A.C., Holzapfel, W., Shortt,
by the increased induction of IL-10 C., Fonden, R., Miller, G.D., Donohue, D.,
Lactobacillus rhamnosus Lr-32 antibiogram
in vitro Playne, M., Crittenden, R., Salvadori, B. &
Amoxicillin S -- L. rhamnosus Lr-32 has shown anti- Zink, R. (2002). Inventory of microorgan-
Ampicillin S inflammatory properties, as demon- isms with a documented history of safe
Ceftazidime R strated through significant protection use in food. Bulletin of the International
Chloramphenicol I against TNBS-induced colitis in an Dairy Federation. 377: 10-19.
Ciprofloxacin R animal model 9. List of taxonomic units proposed for
Clindamycin S QPS status http://www.efsa.europa.eu/
Cloxacillin S REFERENCES EFSA/Scientific_Opinion/sc_op_ej587_
Dicloxacillin S Publications on L. rhamnosus Lr-32 in bold. qps_en.pdf.
Erythromycin S 1.  Ahrné, S., Lönnermark, E., Wold A.E., 10. Foligne, B., Nutten, S., Grangette,
Åberg, N., Hesselmar, B., Saalman, R., C., Dennin,V., Goudercourt, D., Poiret,
Gentamicin R
Strannegård, I.L., Molin, G. & Adlerberth, S., Dewulf, J., Brassart, D., Mercenier, A.
Imipenem R
I. (2005). Lactobacilli in the intestinal & Pot, B. (2007). Correlation between
Kanamycin R
microbiota of Swedish infants. Microbes in vitro and in vivo immunomodula-
Neomycin R
and infection. 7:1256-1262. tory properties of lactic acid bacteria.
Nitrofurantoin R 2. Collins, M.D., Phillips, B.A. & Zanoni, P. World Journal of Gastroenterology.
Penicillin G S (1989). Deoxy-ribonucleic acid homology 13(2):236-243.
Polymixin B R studies of Lactobacillus casei, Lactobacillus 11. Foligne, B., Zoumpopoulou, G.,
Rifampicin S paracasei sp. nov. subsp. paracasei and Dewulf, J., Ben Younes, A., Chareyre,
Streptomycin R subsp. tolerans and Lactobacillus rham- F., Sirard, J.-C., Pot, B. & Grangette,
Sulfamethoxazole R nosus sp. nov. comb. nov. International C. (2007). A Key Role of Dendritic
Tetracycline I Journal of Systematic Bacteriology. 39, Cells in Probiotic Functionality. PloS
Trimethoprim R 105-108. ONE 2(3): e313. doi:10.1371/journal.
Vancomycin R 3.  Aguirre, M. & Collins, M.D. (1993). pone.0000313.
S = Susceptible (minimum inhibitory Lactic acid bacteria and human clinical
concentration ≤ 4µg/ml) infections. J.  Appl. Bact. 75:95-107.
I = Intermediate (minimum inhibitory
concentration = 8 to 32µg/ml)
4. Gasser, F. (1994). Safety of lactic acid
R = Resistant (minimum inhibitory bacteria and their occurrence in human
concentration ≥ 64µg/ml) clinical infections. Bull. Inst. Pasteur.
Table 1. 92:45-67.

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 Danisco A/S The information contained in this
Edwin Rahrs Vej 38 publication is based on our own
DK-8220 Brabrand, Denmark research and development work
Telephone: +45 89 43 50 00 and is to the best of our knowl-
Telefax: +45 86 25 10 77 edge reliable.
info@danisco.com   Users should, however, conduct
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suitability of our products for their
own specific purposes and the
legal status for their intended use
of the product.
  Statements contained herein
should not be considered as a
warranty of any kind, expressed
or implied, and no liability is ac-
cepted for the infringement of
any patents.
  Regarding Health Claims,
users should conduct their own
legal investigations into national
demands when marketing and
selling a consumer product con-
taining the probiotic described in
this technical memorandum.

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