Branched lactose containing polysacharides and compositions containing them

The present invention concerns a new branched natural soluble polysaccharide comprising a main chain having repeating side chains which are only made of lactose units, possibly substituted. The present invention also concerns the microorganism by which this branched polysaccharide may be obtained and the food composition, the cosmetical composition and the pharmaceutical composition comprising said branched polysaccharide and/or microorganism.

FIELD OF THE INVENTION 
The present invention concerns a new branched polysaccharide, a 
microorganism producing it, the food composition, the pharmaceutical 
composition and the cosmetical composition containing them. 
BACKGROUND OF THE INVENTION 
Biological communication (the possibility for a cell to recognize a 
molecule or another cell) is a central phenomenon in pathological as well 
as in normal states. 
Among the various mechanisms of molecular recognition between cells, and/or 
between cells and molecules, the binding of specific glycosidic structures 
by specialized proteins (lectins) is today considered as a major molecular 
recognition system. 
The lectins may be bound specifically and non-covalently to well-defined 
glycosidic sequences. 
Some lectins are bound, for example, to oligosaccharides which contain 
elevated mannose amounts, to structures carrying sialic acids, or to 
fucosylated glycosides. 
Other lectins can bind .beta.-galactosides and lactose. 
Multigeneric coaggregations exist between oral bacterial cells (such as 
Actinomyces naeslundii or viscosus, Streptococcus mitis or sanguis, 
Fusobacterium nucleatum, Porphyromonas gingivalis, Bacteriodes 
intermedius, etc.) which aggregate and form a network as the dental 
plaque. 
Between these bacterial cells, the interaction is often obtained by a 
non-covalent bond between a .beta.-galactoside lectin on one cell and a 
glycosidic receptor on another cell (ref. 1). 
Most infectious diseases are initiated by the adhesion of pathogenics 
agents (such as Actinomyces naeslundii, Fusobacterium nucleatum, 
Bacteriodes intermedius, Salmonella typhimurium, Vibrio Cholera, 
Campylobacter jejuni, Bacteriodes, Fusobacteria, Clostridia, Shigella, 
Yersinia, and Helicobacter pylori, etc.) to the epithelial cells of the 
mucosa of its host, which allows then the colonisation of the animal 
tissues. 
This adhesion is often obtained by a binding between a .beta.-galactoside 
lectin located at the surface of this pathogeneous agent and a glycosidic 
receptor located at the surface of the epithelial cell (ref. 2). 
Various cells of the immune system (lymphocytes T and B. macrophages, 
neutrophils) are known either to be able to bind .beta.-galactoside 
lectins or to express at their surface such lectins of the galectin 
family. 
In addition, some epithelial cells such as intestinal cells or 
keratinocytes produce these galectins which can also coat Langerhans 
cells, and immunoglobulins such as IgE can specifically bind to galectins 
(ref. 3, 4 and 5). 
STATE OF THE ART 
There have been many prior studies upon polysaccharides produced by 
micro-organisms and, in recent years, there have been several reports of 
studies on the structure of exocellular polysaccharides obtained from 
lactic acid bacteria and on their biological activities. 
A polysaccharide consisting of galactose, glucose and N-acetylgalactosamine 
(2:1:1) is obtained by the strains of Streptococcus thermophilus CNCM 
I-733, CNCM I-734 and CNCM I-735 (ref. 6 and 7); 
a polysaccharide consisting of galactose only is obtained by the strain 
Lactococcus cremoris H414 (ref. 8); 
a polysaccharide consisting of galactose, glucose, rhamnose and phosphate 
(2:2:1:1) is obtained by the strain Lactococcus cremoris SBT 0495 (ref. 
9); 
a polysaccharide consisting of galactose, glucose and rhamnose (5:1:1) is 
obtained by the strain Lactobacillus bulgaricus rr (ref. 10); 
a polysaccharide consisting of glucose, rhamnose, 1-phosphoglycerol and a 
O-acetyl group (3:2:1:0.85) is obtained by the strain Lactobacillus sake 
0-1 (ref. 11). 
On the other hand, other polysaccharides obtained by a few strains of 
Lactobacillus helveticus were studied, but their structural 
characterization were never performed. For example, a polysaccharide of 
unknown structure consisting of glucose and galactose (2:1) used as an 
anti-tumor agent is obtained by the strain Lactobacillus helveticus var. 
jugurti No 851 "FERM BP-66 (FERM-P No 5851)" (ref. 12 and 13). Similarly, 
a polysaccharide of unknown structure consisting of galactose, glucose and 
N-acetylgluccsamine (2.5-3.5:2.5-3.5:1) used in treating inflammation and 
to accelerate bone marrow growth is obtained by the strain Lactobacillus 
helveticus MIKI-010 (ref. 14). 
AIMS OF THE INVENTION 
The present invention aims to provide a new branched polysacharide and/or 
the microorganism producing it, which can be used to inhibit the binding 
between .beta.-galactoside lectins and their receptor(s). 
Another aim of the invention is to provide food compositions comprising 
said polysaccharide and/or microorganism, having improved organoleptic and 
texture properties. 
A further aim of the invention is to provide a pharmaceutical composition 
and/or cosmetical composition, comprising said polysaccharide and/or 
microorganism. 
A last aim of the invention is to provide a polysaccharide which can be 
used as an intermediate product for the production of polymerized 
derivatives of gangliotriose, Sd-a blood group, or sialyl- and 
sulfated-Lewis X. 
DESCRIPTION OF THE INVENTION 
The present invention concerns a new natural soluble branched 
polysaccharide comprising a main chain having repeating side chains which 
are only made of lactose units, possibly substituted. 
According to a preferred embodiment of the present invention, the branched 
polysaccharide corresponds to the following formula: 
##STR1## 
where n&gt;1, Gal=galactose, 
Glc=glucose, 
R.sup.1 =Hydrogen or GalNAc.beta.1 (N-acetylgalactosamine) 
R.sup.2 =Hydrogen, NeuNAc.alpha.2 (N-acetylneuraminic Acid) or HSO.sub.3 
R.sup.3 =Hydrogen or Fuc.alpha.1 (fucose) 
When R.sup.1 =R.sup.2 =R.sup.3 =Hydrogen, the branched polysaccharide is 
characterized by the following physicochemical properties: 
molecular weight higher than 2,000,000, soluble in water and solutions 
containing less than 20% trichloroacetic acid, 
insoluble in alcohol and in acetone, 
neutral property, 
the freeze-dried product is in the form of white powder, 
component sugars and compositional ratio:Glucose:Galactose (1:1.1). 
When R.sup.1 is GalNAc.beta.1 (N-acetyl galactosamine) and R.sup.2 =R.sup.3 
=Hydrogen, the branched polysaccharide is a derivative of the 
gangliotriose determinant which is advantageously obtained from an 
intermediate product (the polysaccharide with R.sup.1 =R.sup.2 =R.sup.3 
=Hydrogen) by the methods described in documents 15 and 16. 
When R.sup.1 is GalNAc.beta.1 (N-acetyl galactosamine), R.sup.2 is 
NeuNAc.alpha.2 (N-acetyl neuraminic Acid) and R.sup.3 is Hydrogen, the 
branched polysaccharide is a derivative of the blood group Sd-a 
determinant, which is advantageously obtained from an intermediate product 
(the polysaccharide with R.sup.1 =R.sup.2 =R.sup.3 =Hydrogen) by the 
methods described in documents 17 and 18. 
When R.sup.1 is Hydrogen R.sup.2 is NeuNAc.alpha.2 (N-acetylneuraminic 
Acid) or HSO.sub.3 and R.sup.3 is Fuc.alpha.1 (fucose), the branched 
polysaccharide is a derivative of the pharmaceutical products sialyl- or 
sulfated- Lewis X described in documents 19 and 20. 
The bindings between Gal .beta. 1-4 Glc and R.sup.1, R.sup.2 are 
advantageously obtained from an intermediate product (the polysaccharide 
with R.sup.1 =R.sup.2 =R.sup.3 =Hydrogen) by the method described in 
document 17. 
The present invention also concerns the microorganism producing the 
branched polysaccharide having R.sup.1 =R.sup.2 =R.sup.3 =Hydrogen. 
Advantageously, said microorganism corresponds to a strain of Lactobacillus 
helveticus, preferably the strain of Lactobacillus helveticus CNCM I-1449. 
A deposit of this microorganism has been made according to the Budapest 
Treaty on Jul. 27, 1994 under acession number CNCM I-1449, at the 
Collection Nationale de Culture de Microorganismes (CNCM), Institut 
Pasteur, 28 rue du Docteur Roux, 75724 IS CEDEX 15, FRANCE. 
The present invention also concerns the food having improved organoleptic 
and texture properties comprising the polysaccharide and/or the 
microorganism according to the invention. 
Preferably, said food composition is a set-style acidified milk or a 
stirred acidified milk. 
Another aspect of the present invention concerns a cosmetic composition 
comprising the polysaccharide and/or the microorganism according to the 
invention. 
According to a preferred embodiment of the invention, said cosmetic 
composition is a cosmetic product intended for buccal hygiene, choosen 
among the group consisting of a tooth paste, tooth gel, mouth rinse, 
chewing-gum and/or tablet. 
According to another preferred embodiment of the present invention, said 
cosmetic composition is a product intended for skin hygiene, choosen among 
the group consisting of a cream, ointment or balsam. 
Another aspect of the present invention concerns a pharmaceutical 
composition comprising the polysaccharide and/or the microorganism 
according to the present invention. 
Advantageously, said composition is a antidiaorrheic product choosen among 
the group consisting of a capsule, syrup, powder and/or tablet. 
A last aspect of the present invention concerns a diagnostic and/or 
analytic device comprising the branched polysaccharide according to the 
invention for the trapping of specific molecules and/or microorganisms.

The compositions comprising the polysaccharide and/or microorganism 
according to the invention are described in the following non limiting 
examples. 
EXAMPLE 1 
Set-Style Acidified Milk. 
Set-style acidifed milk comprising the L. helveticus strain according to 
the invention and two S. thermophilus strains, traditionnaly used for the 
production of a set-style yoghourt, was obtained by the following process. 
To a whole milk comprising 3.7% fats, 2.5% skimmed milk powder was added. 
40 liters of this milk was pasteurized at 92.degree. C. for six minutes, 
homogeneized at 75.degree. C. and 150 bars (two levels) and cooled at a 
temperature around 42.degree. C. 
The freeze-dried S. thermophilus CNCM I-1422, S. thermophilus CNCM I-1424 
and L. helveticus CNCM I-1449 strains were reactived with several 
successive cultures in a sterile MSK medium (skimmed milk powder 
reconstituted at 10%, comprising 0,1% of a commercial yeast extract). 
A deposit of the microorganism has been made according to the Budapest 
Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 
1994, for the Lactobacillus strain at the Collection Nationale de Culture 
de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 
IS CEDEX 15, FRANCE. 
The sterilized milk was inoculated with 1% of the third culture of each S. 
thermophilus strain and with 2% of the second culture of L. helveticus 
strain taken at the medium coagulation stage. 
The milk was incubated at 42.degree. C. and at a pH around 4.65, and then 
cooled at a temperature of 4.degree. C. 
EXAMPLE 2 
Acidified Whey Milk. 
Whey milk comprising the L. helveticus strain acording to the invention and 
two S. thermophilus strains, traditionnaly used for the production of a 
yoghourt, was obtained by the following process. 
A sweet lactoserum powder was reconstituted at 12.5% in water. 
40 liters of this whey was pasteurized at 92.degree. C. for six minutes, 
homogeneized at 75.degree. C. and 150 bars (two levels) and cooled at a 
temperature around 42.degree. C. 
The freeze-dried S. thermophilus CNCM I-1422, S. therrmophilus CNCM I-1424 
and L. helveticus CNCM I-1449 strains were reactived with several 
successive cultures in a sterile MSK medium (skimmed milk powder 
reconstituted at 10%, comprising 0.1% of a commercial yeast extract). 
A deposit of the microorganism has been made according to the Budapest 
Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 
1994, for the Lactobacillus strain at the Collection Nationale de Culture 
de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 
IS CEDEX 15, FRANCE. 
The sterilized milk was inoculated with 1% of the third culture of each S. 
thermophilus strain and with 2% of the second culture of L. helveticus 
strain taken at the medium coagulation stage. 
The whey milk was incubated at 42.degree. C. and at a pH around 4.65, and 
then cooled at a temperature of 4.degree. C. 
EXAMPLE 3 
Stirred Acidified Milk. 
A stirred acidifed milk comprising the L. helveticus CNCM I-1449 strain 
according to the invention and two commercialized S. thermophilus strains, 
traditionnaly used for the production of a stirred yoghourt, was obtained 
by the following process. 
The milk was obtained from a whole milk comprising 3.7% fats, by the 
addition of 2.5% skimmed milk powder. 
40 liters of this milk was pasteurized at 105.degree. C. for two minutes, 
homogeneized at 75.degree. C. and 300 bars (first level) and cooled at a 
temperature around 43.degree. C. 
The lyophilized S. thermophilus CNCM I-1421, S. thermophilus CNCM I-1423 
and L. helveticus CNCM I-1449 strains were reactived with several 
successive cultures in a sterile MSK medium. 
A deposit of the microorganism has been made according to the Budapest 
Treaty on May 18, 1994, for the Streptococcus strains and on Jul. 27, 
1994, for the Lactobacillus strain at the Collection Nationale de Culture 
de Microorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 
IS CEDEX 15, FRANCE. 
The sterilized milk was inoculated with 1% of the third culture of each S. 
thermophilus strain and with 2% of the third culture of L. helveticus 
strain taken at the medium coagulation stage. 
The milk was incubated at 43.degree. C. and at a pH around 4.65, and then 
cooled at a temperature of 4.degree. C. during stirring. 
The following table 5 represents the properties of the obtained products. 
TABLE 5 
______________________________________ 
Example 1 Example 3 
______________________________________ 
Acidification 6 h 7 h 15 
time at pH = 4.65 
pH of the product 
4.34 4.49 
after 1 day at 4.degree. C. 
pH of the product 
4.1 4.3 
after 24 days at 
4.degree. C. 
taste after 24 good taste, very good taste, 
days slightly acid 
aromatic 
smooth texture 
smooth and 
onctuous texture 
______________________________________ 
EXAMPLE 4 
Cosmetic Composition for Buccal Hygiene. 
______________________________________ 
CHEMICAL NAME TRADE NAME % WEIGHT 
______________________________________ 
PHASE A 
PEG-40 Hydrogenated 
Cremophor RH 40 
0.10 
castor oil 
Flavour Strawberry E 2226 
0.04 
Flavour Raspberry 9/022436 
0.10 
PHASE B 
Sodium Cyclamate 
Sodium Cyclamate 
0.10 
Exopolysaccharide 
-- 0.50-5.00 
according to the 
present invention 
Demineralized water 
-- 94.66-99.16 
TOTAL 100 
______________________________________ 
EXAMPLE 5 
Cosmetic Composition for Skin hygiene. 
______________________________________ 
% WEIGHT 
______________________________________ 
OIL PHASE 
BRIJ 721 (Steareth 21) 
4.00 
Cetyl alcoho1 10.00 
Mineral oil 5.00 
Propyl parahydroxybenzoate 
0.02 
WATER PHASE 
CARBOPOL 934 (Carbomer 934) 
0.10 
Sodium hydroxide (solution 
0.10 
at 10%) 
Methyl parahydroxybenzoate 
0.18 
Exopolysaccharide according 
0.50-5.00 
to the present invention 
Demineralized water 75.60-80.10 
TOTAL 100 
______________________________________ 
EXAMPLE 6 
Pharmaceutical composition for anti-diarrheoic usage. 
A pharmaceutical composition was obtained as a capsule which was made with 
gelatine and water, and which contained from 5 to 50 mg of the 
exopolysaccharide according to the present invention. Alternatively, 
powdered tablet formulations can be obtained directly from the acidified 
cultured milks described in the above examples 1, 2 and 3, by 
freeze-drying these fermented milks and whey. 
References 
1. Kolenbrander, P. E., Ganeshkumar, N., Cassels, F. J. & Hughes, C. V., 
Coaggregation: specific adherence among human oral plaque bacteria. The 
FASEB Journal, 7 (1993) 406-413. 
2. Karlsson, K.-A., Animal glycosphingolipids as membrane attachment sites 
for bacteria. Annual Review of Biochemistry, 58 (1989) 309-350. 
3. Hughes, R. C., Mac-2: A versatile galactose-binding protein of mammalian 
tissues. Glycobiology, 4 (1994) 5-12. 
4. Truong, M.-J., Gruart, V., Kusnierz, J.-P., Papin, J.-P., Loiseau, S., 
Capron, A. & Capron, M., Human neutrophils express immunoglobulin E 
(IgE)-binding proteins (Mac-2/.epsilon.BP) of the S-type lectin family: 
role in IgE-dependent activation. Journal of Experimental Medicine, 177 
(1993) 243-248. 
5. Wollenberg, A., de la Salle, H., Hanau, D., Liu, F.-T. & Bieber, T., 
Human keratinocytes release the endogenous .beta.-galactoside-binding 
soluble lectin immunoglobulin E (IgE-binding protein) which binds to 
Langerhans cells where it modulates their binding capacity for IgE 
glycoforms. Journal of Experimental Medicine, 178 (1993) 777-785. 
6. Doco, T., Fournet, B., Carcano, D., Ramos, P., Loones, A., Piot, J.-M. & 
Guillochon, D., Polysaccharide, application comme agent epaississant et 
comme agent anti-tumoral. Demande de brevet europeen, EUR 331 564, 
06.09.1989. 
7. Doco, T., Wieruszeski, J.-M., Fournet, B., Carcano, D., Ramos, P. & 
Loones, A., Structure of an exocellular polysaccharide produced by 
Streptococcus thermophilus. Carbohydrate Research, 198 (1990) 313-321. 
8. Gruter, M., Leeflang, B. R., Kuiper, J., Kamerling, J. P. & 
Vliegenthart, J. F. G., Structure of the exopolysaccharide produced by 
Lactococcus lactis subspecies cremoris H414 grown in a defined medium or 
skimmed milk. Carbohydrate Research, 231 (1992) 273-291. 
9. Nakajima, H., Hirota, T, Toba, T., Itoh, T. & Adachi, S., Structure of 
the extracellular polysaccharide from slime-forming Lactococcus lactis 
subsp. cremoris SBT 0495. Carbohydrate Research, 224 (1992) 245-253. 
10. Gruter, M., Leeflang, B. R., Kuiper, J., Kamerling, J. P. & 
Vliegenthart, J. F. G., Structural characterization of the 
exopolysaccharide produced by Lactobacillus delbruckii subspecies 
bulgaricus rr grown in skimmed milk. Carbohydrate Research, 239 (1993) 
209-226. 
11. Van den Berg, D. J. C., Ledeboer, A. M., Robijn, G. W. & Vreeker, R., 
Lactobacillus sake like strains, production and use of their 
exopolysaccharides. International Patent Application PCT No WO 94/12656, 
Jun. 9, 1994. 
12. Tsuchiya, F., Miyazawa, K., Kanbe, M., Oda, M. & Ebisawa, N., High 
molecular polysaccharide MPS-80. U.S. Pat. No. 4,396,763, Aug. 2, 1983. 
13. Oda, M., Hasegawa, H., Komatsu, S., Kambe, M. & Tsuchiya, F., 
Anti-tumor polysaccharide from Lactobacillus sp. Agricultural and 
Biological Chemistry, 47 (1983) 1623-1625. 
14. Yamamoto, Y., Polysaccharide NPS, method for making it and uses 
thereof. Laid-open patent application from the Japanese Patent Office No 
5-186501, Jul. 27, 1993. 
15. Lutz M. S, Jaskiewica E., Darling D. S., Furukawa K., Young W. W. Jr., 
Cloned beta-1, 4 N-acetylgalactosaminyltransferase synthesizes G-A2 as 
well as gangliosides G-M2 and G-D2: G-M3 synthesis has priority over G-A2 
synthesis for utilization of lactosylceramide substrate in vivo. Journal 
of Biological Chemistry, 269 (1994) 29227-29231. 
16. Yamashiro S., Haraguchi M., Furukawa K., Takamiya K., Yamamoto A., 
Nagata Y., Lloyd K. O., Shiku H., Furukawa K., Substrate specificity of 
beta-1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA 
transfected cells: G-M2-G-D2 synthase efficiently generates asialo-G-M2 in 
certain cells, Journal of Biological Chemistry 270 (1995) 6149-6155. 
17. Ichikawa, Y., Lin, Y.-C., Damas, D. P., Shen, G.-J., Garcia-Junceda, 
E., Williams, M. A., Bayer, R., ketcham, C., Walker, L. E., Paulson, J. C. 
& Wong, C.-H., Chemical-enzymatic synthesis and conformational analysis of 
sialyl Lewis X and derivatives. Journal of the American Chemical Society, 
114 (1992) 9283-9298. 
18. Smith P. L., Lowe J. B., Molecular cloning of a murine 
N-acetylgalactosamine transferase cDNA that determines expression of the T 
lymphocyte-specific CT oligosaccharide differentiation antigen, Journal of 
Biological Chemistry, 269 (1994) 15162-15171. 
19. Hodgson, J., Carbohydrate-based therapeutics. Bio/Technology, 9 (1991) 
609-613. 
20. Yuen, C. T., Bezouska, K., O'Brien, J., Stoll, M., Lemoine, R., 
Lubineau, A., Kiso, M., Hasegawa, A., Bockovitch, N. J., Nicolaou, K. C. & 
Feizi, T., Sulfated blood group Lewis.sup.a. A superior oligosaccharide 
ligand for human E-selectin. Journal of Biological Chemistry, 269 (1994) 
1595-1598. 
21. Neeser, J.-R. & Schweizer, T. F., A quantitative determination by 
capillary gas-liquid chromatography of neutral and amino-sugars (as 
O-methyloxime acetates), and a study on hydrolytic conditions for 
glycoproteins and polysaccharides in order to increase sugar recoveries. 
Analytical Biochemistry, 142 (1984) 58-67.