WO2004093899A1

WO2004093899A1 - Immunomodulatory product obtained from a bifidobacterium culture and compositions containing the same

Info

Publication number: WO2004093899A1
Application number: PCT/FR2004/000875
Authority: WO
Inventors: Valérie PETAY; Francis Lecroix; Emmanuel Perrin; Charles Gontier; Jean-Pierre Blareau; Marie-Bénédicte ROMOND; Elisabeth Singer; Marie-Françoise ODOU; Catherine Demailly-Mullie
Original assignee: Compagnie Gervais Danone
Priority date: 2003-04-16
Filing date: 2004-04-08
Publication date: 2004-11-04
Also published as: FR2853908A1; EP1615657A1; FR2853908B1

Abstract

The invention relates to an immunomodulatory product obtained from a Bifidobacterium culture, to the use thereof, especially as a medicament or a food ingredient, and to pharmaceutical or food compositions containing the same.

Description

IMMUNOMODULATOR PRODUCED FROM A BIFIDOBACTERIUM CULTURE AND COMPOSITIONS CONTAINING THE SAME

The present invention relates to an immunomodulatory product obtained from a culture of Bifidobacterium, to its use, in particular as a medicament or food ingredient, as well as to pharmaceutical or food compositions containing it.

The genus Bifidobacterium belongs to the family of Actinomycetaceae; it combines bacilli with Qram positive, strict anaerobes, fermenting glucose by the way of fructose 6-phosphate phosphoketolase. Their optimal growth pH is between 6 and 7, and their optimal growth temperature is between 37 and 46 ° C.

Bifidobacteria are part of the normal human intestinal flora, and are known to have many beneficial health effects. It is known in particular that breastfed infants, who have an intestinal flora in which bifidobacteria predominate, are more resistant to infections and in particular have a lower risk of diarrhea than infants fed with conventional industrial milk preparations.

The role of bifidobacteria in this increased resistance to infection has not been fully elucidated. Different studies indicate that they have an immunomodulatory power which would imply polysaccharide substances associated with the bacterial wall, or secreted by the bacteria during anaerobic fermentation. Gomez et al., (FEMS Microbiol. Lett., 1988, 56, 47-52) describe the immunomodulatory effect of exocellular fractions rich in polysaccharides produced by Bifidobacterium adolescentis; patent application FR 2 652 590 describes an immunopotentiating exopolymer of polysaccharide nature produced by a strain of the continuum Bifidobacterium infantis longum; Honoso et al. (Biosci. Biotech. Biochem., 1997, 61, 312-316 and Bioscience Microflora, 1998, 17, 97-104) describe immunopotentiating polysaccharides produced by different species of Bifidobacterium. The immunomodulatory action of bifidobacteria is also manifested by the regulation of the intestinal micro flora, in particular at the expense of the development of pathogenic bacterial species. Romond et al. (Anaerobe, 1997, 3, 137-143 and J. Dairy Sci., 1998, 81, 1229-1235) thus describe fractions rich in glycoproteins, produced by brief Bifidobacterium under anaerobic fermentation conditions, and induce in vivo a regulatory effect on the intestinal microflora.

There are thus on the market many products fermented by bifidobacteria, possibly associated with other lactic acid bacteria, and whose ingestion allows to benefit from the immunomodulatory effects of bifidobacteria and their fermentation products.

However, and in the particular case of infant nutrition, these have the disadvantage of being too acidic and of presenting, in particular in the case of powdered products, a non-homogeneous appearance after reconstitution, due to the coagulation of milk proteins by the acidity generated during fermentation. They are therefore sometimes badly accepted by the child and by the mother.

In order to remedy these drawbacks, there has already been proposed in international application WO 01/01785, a process for the preparation of an immunostimulating milk product by bioconversion, without fermentation and therefore without acidification of the final product, from a milk substrate to using bifidobacteria, and in particular the brief Bifidobacterium strain, deposited according to the Budapest Treaty, on May 31, 1999, under number 1-2219 with the CNCM (National Collection of Cultures of Microorganisms) held by the Institute Pasteur, 25 rue du Docteur Roux, in Paris. However, the preparation process described in this international application does not make it possible to prepare food products other than milk products and requires implementation conditions which are restrictive from an industrial point of view, in particular the maintenance of aerobic culture conditions, maintaining the culture medium at an osmotic pressure corresponding to 0.93 to 0.97 of water activity (AW), and / or maintaining the culture medium at a temperature between 40 and 48 ° C.

Furthermore, Clostridium perfringens is an anaerobic, Gram-positive, spore-forming bacterium which is very widespread in the environment (soil, water, air, dust) but is also common in humans and animals (flora of the intestine, vagina, upper airways ...). The bacteria Clostridium perfringens are responsible for the release of toxins and enzymes in the digestive tract which cause tissue damage. The production of these different toxins is associated with sporulation. Spores persist in soil, sediments and surfaces exposed to human and animal faecal pollution and their presence in water is a criterion for faecal contamination.

Clostridium perfringens is responsible for gas gangrene, food poisoning and necrotizing enteritis.

Although other germs are involved in the occurrence of myonecrosis, Clostridium perfringens remains the main agent of gas gangrene. It is generally post-traumatic (following accidents) but can also be post-operative (especially in visceral septic surgery), purely medical (following a muscular injection) or even spontaneous (on colon cancer). Gas gangrene is fatal in 25 to 60% of cases.

Clostridium perfringens is involved in 10 to 15% of food poisoning. Contamination occurs through ingestion of foods such as meat, milk, fruits and vegetables which are frequently contaminated with this bacteria. Once ingested, if the conditions are appropriate, the body produces toxins in the gastrointestinal tract and cause symptoms which are characterized, for the common form of infection, by intense intestinal cramps, acute abdominal pain, diarrhea, nausea, vomiting, and fever. Clostridium perfringens is also responsible for a severe digestive condition called necrotizing enteritis, especially in young children or newborns. In the absence of appropriate treatment, it is followed by peritonitis or shock which is often fatal, death resulting from infection and necrosis of the intestine and resulting septicemia. It may therefore be advantageous to be able to have an immunomodulatory product making it possible to control the proliferation of Clostridium perfringens.

It is therefore in order to remedy all the drawbacks presented by the products described in the prior art and to provide an immunomodulatory product which can be incorporated into any type of food product or be used for the preparation of pharmaceutical compositions. immunomodulatory, that the Inventors have developed which is the subject of the Invention.

The inventors have also set themselves the goal of providing a food or pharmaceutical composition having a regulatory effect on the intestinal microflora, in particular at the expense of the development of pathogenic bacterial species, in particular Clostridium perfringens.

The present invention therefore has for first object an immunomodulatory product characterized in that it is obtained according to a preparation process comprising the following steps: - seeding and incubation, under aerobic or anaerobic conditions, preferably anaerobic, and at a temperature of between 30 and 40 ° C, of Bifidobacterium comprising at least the brief Bifidobacterium strain 1-2219 in an aqueous substrate having a pH of between 6 and 8 approximately, and comprising at least the following ingredients: i) permeate whey, ii) a whey protein hydrolyzate, iii) lactose,

- elimination of Bifidobacterium from the aqueous substrate;

- Ultrafiltration of the aqueous substrate on filtration membranes having a cutoff threshold between 100 and 300 kDa to obtain a concentrated retentate;

- dehydration of the concentrated retentate, preferably by lyophilization;

- dissolving the dehydrated retentate in a buffer; - column exclusion chromatography on a column having an exclusion threshold of 600 kDa from the retentate solution;

- recovery of the filtered fraction at the end of the chromatography which constitutes the immunomodulatory product.

The filtered fraction obtained by implementing the process in accordance with the invention has immunomodulatory properties; it makes it possible in particular to stimulate the proliferation of Bifidobacterium and to decrease the population of Clostridium perfringens within the intestinal flora. According to the invention, the whey permeate used in the composition of the aqueous substrate may be in the form of a powder, obtained by ultrafiltration of whey, after drying (by spray or by any other drying technique), of the fraction liquid, demineralized or not, which crosses the membrane during the ultrafiltration of the whey.

The whey protein hydrolysates which can be used in the context of the present invention can be obtained by the methods usually used for the preparation of protein hydrolysates, in particular by enzymatic hydrolysis of whey proteins using proteases such as trypsin, chymotrypsin, etc. Many concentrates or isolates of serum proteins as well as hydrolysates of whey proteins, suitable for the implementation of the invention are known in themselves and available commercially.

Before its use, the aqueous substrate is preferably filtered on membranes, such as for example on polyethersulfone membranes, having a cut-off threshold of between 100 and 300 kDa, then the autoclave permeate at a temperature of approximately 120 ° C. for approximately 30 minutes in order to avoid any undesirable bacterial contamination of the culture medium.

Before use, the pH of the aqueous substrate can be adjusted to the desired value using any basifying agent conventionally used by those skilled in the art such as, for example, soda or potash.

The Bifidobacterium are preferably sown in the aqueous substrate at a rate of 1.10 ⁴ to 4.10 ⁹ colony forming units (CFU) per ml of substrate. This seeding can be carried out for example by adding to the aqueous substrate, in suitable proportions, a frozen concentrate of Bifidobacterium, or a preculture on medium allowing the growth of bifidobacteria.

According to a preferred embodiment of the invention, the pH of the aqueous substrate is maintained at a value between approximately 6 and 8 throughout the incubation period, and even more preferably between 6.5 and 7.5. Maintaining the pH is preferably achieved by continuous neutralization of the aqueous substrate using an alkalizing agent as described above or by a dilute ammonia solution (preferably half). According to a preferred embodiment of the invention, the temperature of the substrate is maintained at a value of between 37 and 40 ° C. for the entire duration of the incubation, the latter generally being between 10 and 20 hours. According to a preferred embodiment of the process according to the invention, the ingredients of the aqueous substrate are present in the following amounts: i) whey permeate: from 3 to 80 g approximately and even more preferably from 40 to 60 g approximately, ii ) whey protein hydrolyzate: from 2 to 80 g approximately and even more preferably from 5 to 15 g approximately, iii) lactose: from 5 to 50 g approximately and even more preferably from 10 to 30 g approximately, these quantities being given by liter of said aqueous substrate. According to a particular embodiment of the invention, the aqueous substrate can also comprise at least one additional ingredient chosen from yeast extracts, buffer salts and cysteine hydrochloride.

When the aqueous substrate comprises a buffer salt, this is preferably chosen from sodium dihydrogen phosphate and potassium dihydrogen phosphate and then preferably represents from 0.5 to 5 g approximately and even more preferably from 1.5 to 3 g approximately per liter of aqueous substrate.

When the aqueous substrate comprises a yeast extract, this then preferably represents from 0.5 to 5 g approximately, and even more preferably from 1.5 to 3 g approximately per liter of aqueous substrate.

When the aqueous substrate comprises cysteine hydrochloride, the latter then preferably represents from 100 to 500 mg approximately and even more preferably from 200 to 400 mg approximately per liter of aqueous substrate.

At the end of the incubation period, the elimination of Bifidobacterium from the culture medium can be carried out for example by microfiltration, or by centrifugation of the aqueous substrate. According to a preferred embodiment of the invention, the elimination of Bifidobacterium from the medium of culture is carried out by centrifugation of the aqueous substrate, for example at a speed of 3000 g for a period of approximately 1 hour.

According to a preferred embodiment of the invention, the method further comprises, after the step of eliminating Bifidobacterium, an additional step of destroying the residual enzymatic activities contained in the aqueous substrate after incubation, for example by heat treatment of it at a temperature of about 75 ° C for about 3 minutes.

The step of ultrafiltration of the aqueous substrate is preferably carried out on polyethersulfone membranes, at a temperature below about 60 ° C. At the end of the ultrafiltration step, the concentrated retentate thus obtained is preferably washed several times, for example with permuted water before being finally reconcentrated before being dehydrated, for example by lyophilization.

The buffer used for dissolving the dehydrated retentate is preferably chosen from buffers having a pH between 6 and 8 such as for example the Tris buffer adjusted to the desired pH by addition of hydrochloric acid.

The nature of the gels which can be used to carry out the exclusion chromatography is not critical as long as they have an exclusion threshold of 600 kDa. By way of gel, it is possible in particular to use gels composed of dextran and crosslinked agarose such as the product sold under the trade name Superdex® 200 by the company Amersham Biosciences.

The salts present in the filtered fraction can also optionally be removed by ultrafiltration on a membrane having a cutoff threshold of approximately 10 kDa.

Finally, the filtered fraction constituting the immunomodulatory product can be used directly or frozen or lyophilized for conservation and subsequent use.

This filtered fraction consists of a complex of polysaccharides and proteins in which the carbohydrate fraction represents from 10 to 20% by weight approximately, the protein fraction representing approximately from 80 to 90% by weight relative to the total weight of said complex.

According to the invention, the carbohydrate fraction of the filtered fraction has the following monosaccharide composition (expressed in molar ratios compared to rhamnose): galactose: 11 to 14; mannose: 2 to 5; glucose: 3 to 6; N-acetyl galactosamine: 2.5 to 4.5; N-acetyl glucosamine 0.5 to 1.5; neuraminic acid: 4.5 to 6.5 and rhamnose: 1.

The subject of the invention is also the immunomodulatory product obtained according to the method described above as a medicament, and in particular as an immunomodulatory medicament.

Another object of the invention is a pharmaceutical composition characterized in that it contains, as active principle, at least one immunomodulatory product obtained according to the method described above, and at least one pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” means any support which, while retaining the immunomodulatory product obtained according to the process according to the invention, its properties, particularly its immunomodulatory properties, makes it possible to convey said product. The pharmaceutical composition according to the invention can be in any dosage form desired for oral administration to humans or animals, such as for example in liquid form for a syrup or a solution, a spray, or in the form solid, for example a powder, a tablet, a capsule, a capsule, a powder spray, in their various forms, immediate or programmed release, a gum, a paste, granules, or in any other form suitable for administration by orally.

The immunomodulatory product obtained according to the process according to the invention can also be incorporated, as an ingredient, in food compositions. Consequently, the subject of the invention is also a food composition, characterized in that it contains, as an ingredient, at least one immunomodulatory product obtained according to the process according to the invention.

Such food compositions can be intended for human or animal consumption and can in particular be in the form of a milk or not preparation, fermented or not, of animal or vegetable origin, including in particular infant formulas or for adults and seniors, and in particular in the form of infant formula, liquid or powdered milk, products fresh, cereals, cookies (fodder), small jars, desserts, etc. or even in the form of food or dietetic products for adults, including hospital products, or nutritional supplements.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of preparation of the immunomodulatory product in accordance with the invention, as well as to the appended figures in which:

- Figure 1 shows the chromatogram obtained after injection on a column packed with a Superdex® 200 gel of a culture medium fermented for 15 hours with the strain

Bifidobacterium brief CNCM 1-2219 (absorbance in millivolts as a function of time elapsed in minutes);

- Figure 2 compares the chromatograms obtained after injection on a column packed with a Superdex® 200 gel of a culture medium fermented with the brief Bifidobacterium strain

CNCM 1-2219 or by the short strain B. CFPL (Collection of the Faculty of Pharmacy of Lille) C7 (absorbance in millivolts as a function of the time elapsed in minutes);

- Figure 3 shows an enlargement of Figure 2. EXAMPLE 1: PREPARATION OF AN IMMUNOMODULATOR PRODUCT OBTAINED BY CULTURE OF BIFIDOBACTERIUM

A culture medium is prepared containing the following ingredients:

- 50 g / 1 of whey permeate,

- 10 g / 1 of whey protein hydrolyzate, - 20 g / 1 of lactose,

- 2 g / 1 yeast extract,

- 2.5 g / 1 of potassium dihydrogen phosphate,

- 0.3 g / 1 of cysteine hydrochloride.

The culture medium is ultrafiltered on Centramate® cassettes sold by the company PALL, fitted with polyethersulfone membranes having a cut-off threshold of 200 kDa and the permeate is autoclave for 30 minutes at 120 ° C. The pH of the culture medium is then adjusted to a value of 6.5 using a solution of ammonia diluted to a quarter.

The culture medium is then inoculated with the bifidobacteria at a rate of 6% o (v / v) of a frozen concentrate of the short Bifidobacterium strain CNCM 1-2219 containing 5.10 ¹⁰ CFU of bifidobacteria per ml of frozen concentrate. The initial population of bacteria is 3.10 ⁸ CFU of bifidobacteria per ml of culture medium. Bifidobacteria are cultivated anaerobically, at a temperature between 37 and 40 ° C. During the culture, the pH of the culture medium is regulated at 6.5 using a solution of ammonia diluted to a quarter. The culture time is 15 hours, and the population of Bifidobacterium at the end of the culture is approximately 2.10 CFU per ml of culture medium.

At the end of culture, the bacteria are eliminated from the fermented culture medium by centrifugation for 1 hour at 3000 g. The residual enzymatic activities contained in the centrifugation supernatant are destroyed by a thermal treatment for 3 minutes at 75 ° C.

The supernatant is ultrafiltered on Centramate® cassettes sold by the company PALL, fitted with polyethersulfone membranes having a cutoff threshold of 300 kDa at a temperature of approximately 40 ° C. It is thus concentrated 3 times, then washed 3 times with deionized water. During the last wash, a 7-fold concentration of the part retained by the membrane is operated. This gives a concentrate called retentate. The retentate is dehydrated by lyophilization, then taken up in a Tris NaCl buffer at pH 8.

1) Study of the composition of the retentate obtained

The composition of the retentate is studied by exclusion chromatography.

To do this, 25 μl of retentate is injected at a flow rate of 0.6 ml per minute on a column of Superdex® 200 gel sold by the company Amersham Biosciences and having an exclusion threshold of 600 kDa, coupled to a UV detector at diode array (200-300 nm). The signal integration is carried out using the KromaSystem® 2000 software sold by the company Kontron Instruments. Two fractions are thus separated: a fraction excluded from the gel is eluted after 12.5 minutes at from the injection and a filtered fraction is eluted from 16 to 32 minutes from the injection.

The results obtained on the retentate after 15 hours of fermentation are reported in the appended FIG. 1 which represents the absorbance (in millivolts) as a function of the time elapsed (in minutes) since the injection of the retentate on the column.

These results represent a typical chromatogram showing the excluded fraction and the filtered fraction of the retentate thus analyzed.

Figures 2 and 3 represent the chromatograms obtained after analysis of a retentate from a culture carried out as described above and a retentate from a culture carried out under the same conditions but with a different strain of bifidobacteria (B. brief CFPL C7).

FIG. 2 represents the absorbance (in millivolts) as a function of the time elapsed (in minutes) since the injection on the column of retentates corresponding to the two cultures carried out respectively with the strain B. brief CNCM 1-2219 and with the strain B brief CFPL C7. These results show that, unlike the retentate of the culture carried out with the short B. strain CNCM I-2219, the chromatogram of the retentate of the culture carried out with the short B. strain CFPL C7 shows no peak corresponding to the excluded fraction. and the filtered fraction of the retentate. On the other hand, FIG. 3 which represents an enlargement of FIG. 2, shows that it is necessary to considerably enlarge the absorbance scale of FIG. 2 in order to show the excluded fraction and the filtered fraction of the retentate corresponding to the culture carried out with strain B. brief CFPL C7. These results thus demonstrate that the short strain B. CFPL C7 does not have the potential for production of active principles that represents the short strain B. CNCM 1-2219. 2) Preparation of the filtered fraction of the retentate

The concentrated retentate, previously taken up in a Tris NaCl buffer pH 8, is subjected to preparative chromatography. The separation is carried out by chromatography on a column of Superdex® 200 gel 50 mm in diameter and 100 cm in height, fed at a flow rate of 5 ml per minute and having an exclusion threshold of 600 kDa. The fractions are collected by 10 ml and their absorbance is measured at 280 nanometers.

Two fractions are thus separated: - a fraction excluded from the gel with a molecular weight greater than 600 kDa (retention time from 130 to 180 minutes +/- 10%),

- a filtered fraction of molecular weight between 200 and 600 kDa (retention time from 187 to 370 minutes +/- 10%). The salts are removed from the filtered fraction or active part by ultrafiltration using a membrane having a cutoff threshold of 10 kDa.

The filtered fraction is concentrated at the 3 ^ee washing so as to return to its level of concentration in the retentate. This fraction can then be stored in frozen or lyophilized form. The excluded fraction can also be kept in the same way.

EXAMPLE 2 ANALYSIS OF THE CARBOHYDRATE COMPOSITION OF THE ACTIVE PART (FILTERED FRACTION PREPARED FROM A BIFIDOBACTERIUM CULTURE

The lyophilized powder of filtered fraction as prepared above in Example 1 (10 mg) is taken up in pure anhydrous hydrazine (200-300 microliters). The mixture is incubated overnight at 110 ° C., dried under a stream of nitrogen and taken up in 1 ml of water. The solution is then fractionated by gel permeation on a column

Fractogel TSK, sold under the reference HW40 by the company Merck and the elution is carried out in water. The presence of sugars in the various fractions collected is sought by the orcinol method and by thin layer chromatography. The results obtained (not shown) show that the majority of the sugars are in the fraction not retained on the gel (> 10 kDa) and migrate shortly after thin layer chromatography. The carbohydrate part of the filtered fraction therefore constitutes a polysaccharide with a molar mass greater than 10 kDa. The carbohydrate part of the filtered fraction represents approximately 12% to 16% by mass thereof.

The molar composition of the carbohydrate fraction of the active part is determined by gas chromatography after methanolysis using a mixture of 0.5 M hydrochloric acid and methanol for 24 hours at 80 ° C.

A sample of the filtered fraction (Sample FI), coming from a test carried out according to Example 1, is analyzed.

By way of comparison, the same analyzes were carried out on a sample where the short Bifidobacterium strain CNCM 1-2219 was replaced by the brief Bifidobacterium strain CFPL C7 (Sample C7) cultivated under the same conditions, as well as on a sample where the brief Bifidobacterium strain CNCM I-2219 was grown on a lactose medium not in accordance with the invention (Lactose medium sample: ML) of following composition:

- 60 g / 1 of lactose,

- 2 g / 1 yeast extract,

- 0.3 g / 1 of cysteine hydrochloride.

The following extraction and purification steps being comparable in all respects to those described above.

The molar ratios of given monosaccharides (expressed relative to rhamnose) obtained for the filtered fraction of these different samples are grouped in Table I below:

TABLE I

*: comparative sample not forming part of the invention It can be seen that the distribution of the sugars of the sample FI is different from those of the sample C7 corresponding to the culture of a strain of Bifidobacterium different from the brief strain Bifidobacterium CNCM 1-2219 on a medium in accordance with the invention and that of the ML sample corresponding to the brief Bifidobacterium strain CNCM 1-2219 cultivated on a lactose medium not in accordance with the invention.

In particular, the FI sample contains more galactose and glucose than the C7 and ML samples. EXAMPLE 3 STUDY OF THE IMMUNOMODULATORY ACTIVITY OF THE FILTERED FRACTION OF THE RETENTAT

1) Effect of the filtered fraction on the intestinal flora of the mice

The effect of the filtered fraction (active part), obtained according to the method described above in Example 1, on the evolution of the intestinal flora of the mice was studied.

The test was carried out on C3H male mice which are second generation mice from a line of axenic animals, in which an adult human intestinal flora is implanted (from the CDTA, Center for Distribution, Typing & Archiving of animals, CNRS, Orleans, France).

The mice are kept in isolators to avoid any modification of their new intestinal flora. Upon receipt, the mice are 8-10 weeks old. An adaptation time of at least 2 weeks is left to the mice after reception to remove any stress due to transport and for them to acclimatize to their new environment.

During this entire adaptation period and until the start of the experiment, these mice have sterile water as a drink.

Throughout the duration of the experiment, the food used as a food base is a standard diet of RO3 granules sold by the company UAR, sterilized by irradiation, containing 25% of proteins, 49.8% of carbohydrates, 5% of lipids and 4 % cellulose.

At the age of 12 weeks, start of the experiment, several groups are formed, a group of mice which will receive, during the 15 days of the test, the filtered fraction of the brief Bifidobacterium metabolites CNCM 1-2219 (active part) at the place water from baby bottles, 6 ml per day and per mouse, while two other groups respectively receive the excluded fraction or continue to receive water (control group).

A daily change of the bottles is carried out in order to avoid any modification of the composition of the products to be tested by bacterial proliferation. Feces are collected for analysis before treatment (T0), and on the ^7th and 15 ^th day during the administration of the product (T7 and T15). Each product is tested on a batch of at least 6 mice and the evolution of the bacteria Bifidobacterium and Clostridium perfringens is monitored in the intestinal flora of the mice.

The stool samples are taken in sterile hemolysis tubes and are weighed aseptically then diluted in pre-reduced Ringer's solution diluted to a quarter and supplemented with cysteine hydrochloride (0.3 g / 1), so as to obtain dilutions to the tenth ranging from 10 ^"1 to 10 ^{" 4} . The content is finally deposited at the rate of 100 μl per dish, on different culture media contained in petri dishes:

- Beerens medium (Be) (composed of 35 g / 1 of Columbia agar base, 5 g / 1 of glucose, 0.3 g / 1 of cysteine hydrochloride, 0.5% propionic acid and adjusted to pH 5 ) for finding and counting Bifidobacterium, after spreading dilutions ranging from 10 ^" to 10 ^" ;

- Columbia medium (CC) (composed of 35 g / l of Columbia base (Difco), 15 g / l of agar; adjusted to pH 7.3 and autoclave 15 minutes at 120 ° C) for the search and counting of Clostridium spores, after spreading of heated dilutions ranging from 10 ^"1 to 10 ^{" 2} .

The search and the counting of Bifidobacterium is carried out on the pre-reduced medium Be inoculated directly while the spores of Clostridium perfringens are sought after heating dilutions at 75 ° C for 10 minutes and inoculation on agar CC supplemented with glucose (5 g / 1) , in cysteine hydrochloride (0.3 g / 1) and in horse blood (5% v / v, sold by the company Eurobio).

The spreading is carried out using sterile glass beads on the indicated media and the reading of the media is carried out after 5-7 days of anaerobic incubation at 37 ° C. The identification of the bacteria is carried out on the one hand after description of the colonies obtained on each of the media and on the other hand using a Gram stain.

The results concerning the evolution of Bifidobacterium and

Clostridium perfringens in the feces of mice are reported in Tables II and III respectively: TABLE II

*. p <0.025

TABLE III

**: p <0.05

In these tables, the results obtained are expressed as an average and standard deviation of the log of the number of CFU / g of stool, and n is the number of mice used for each product and days tested (comparison of rank by test of

Wilcoxon for paired samples), the asterisks indicate the significant results compared to time T0. In these tables, the figure mentioned in brackets corresponds to the number of mice in which the bacteria in question are present above the detection threshold. The results corresponding to the unseeded medium are similar to the value obtained at T0 and remain constant over time during the 15 days of the experiment (results not shown in the tables).

These results show that the administration of any one of the two fractions (excluded or filtered) actually leads to an increase in Bifidobacterium but that only the filtered fraction is effective in causing a decrease in the population of Clostridium perfringens within the flora. intestinal mice. 2) Effect of the filtered fraction on the expression of galectins 1 and 3

The measurement of the expression of galectins-1 and -3 is carried out on the mice with adult human flora described above.

At the age of 12-14 weeks, the start of the experiment, two groups of mice are formed, a group of mice which will receive, throughout the duration of the experiment, the filtered fraction containing the brief Bifidobacterium metabolites CNCM 1-2219 instead of baby bottle water, 10 ml per day and per mouse while another group of mice will continue to receive water (control group). 21 ^th day, the mice are sacrificed to carry a biological study on the expression of galectines- 1 and -3, evaluated by assaying the mRNA encoding these galectins by quantitative RT-PCR (Polymerase Chain Reaction).

The modification of the expression of galectins-1 and -3 in the lungs constitutes an indicator of the effect of the metabolites produced by the brief Bifidobacterium strain CNCM 1-2219 contained in the filtered fraction.

Galectins-1 and -3 are measured in the lungs of mice having been sacrificed as described above.

To do this, a fraction of lung (a lobe) is removed sterile from each mouse. The lobe thus removed is then placed in a petri dish previously treated for 24 hours with a sterile solution of water and

DEPC (DiEthyl PyroCarbonate, sold by the company Sigma) at 1/1000. The organ lobe is then perfused a first time with PBS buffer using a syringe and a sterile needle. The lobe is then transferred to another box of

Petri dish containing 200 microliters of PBS supplemented with 50 units of RNase inhibitor (sold by the company Applied Biosystems) and is again perfused with this solution. The lobe is finally cut into slices less than 5 mm which are placed in a Biopur® tube (Eppendorf) containing 0.5 ml of an RNA stabilization solution, sold under the reference RNALater® by the company Qiagen, then stored in the freezer at - 20 ° C until analysis. a) Extraction of total RNA from the lungs

Total RNA is extracted from the tissues after cell lysis according to the protocol for using the RNeasy protect® extraction kit sold under the reference 7126 by the company Qiagen. To do this, the stabilization solution is removed and a tungsten ball treated with DEPC as well as 600 microliters of a lysis solution (RLT buffer contained in the kit) are added. The sample is then ground using a RETSCH MM2000 mill, then centrifuged at 13,000 g for 3 minutes at 4 ° C. The supernatant is then placed in a 1.5 ml tube containing 600 microliters of 70% ethanol and then homogenized. Part of this solution (700 microliters) is then placed on a filtration column which allows the RNA to be hung up, to be centrifuged at 8000 g for 1 minute at 4 ° C. After emptying the collecting tube, 700 microliters of a washing buffer (buffer RW1 contained in the kit) are added to the column which will be centrifuged at 8000 g for 1 minute at 4 ° C. The collecting tube is again emptied and 500 microliters of a second washing buffer containing 70% ethanol (RPE buffer contained in the kit) is added to the column to be centrifuged at 8000 g for 1 minute at 4 ° vs. This last step is repeated but by centrifuging at 13,000 g for 2 minutes at 4 ° C. Finally, 30 microliters of an elution buffer allowing the RNAs to be detached from the column (RNase free buffer contained in the kit) are added to the column. The eluate is then centrifuged at 8000 g for 1 minute at 4 ° C after having been incubated at room temperature for 5 minutes. The elution step is again carried out and the samples thus obtained are quickly frozen at -80 ° C.

An amount equivalent to one unit of DNase, sold by the company Boehringer, is added to each sample which is then incubated in a water bath at 37 ° C for 15 minutes.

In order to assess the amount of total RNA in the samples, they are diluted ^1/8 in sterile water which is placed in a microcuvette allowing the absorbance to be read at 260 nm on a UV spectrophotometer. Genquant reference, sold by the company Pharmacia. To obtain the total RNA concentration of the samples, the value of the optical density (OD) thus obtained is multiplied by the dilution factor and by 40 (one unit of OD = 40 micrograms / ml of RNA). b) Quantitative RT-PCR using the Taq Man® methodology

Quantitative RT-PCR is performed using the qPCR ™ Core Kit sold by the company Eurogentec in a reference thermal cycler Abi Prism 7700 (Sequence Detection System, Applied Biosystems) sold by the company Perkin Elmer. The reaction mixture summarized in the following Table IV is carried out:

TABLE IV

* product supplied in the qPCR Core Kit

The reaction mixture is subjected to the following program: 10 minutes at 65 ° C to remove secondary structures from RNA, 30 minutes at 42 ° C to activate reverse transcriptase, 10 minutes at 95 ° C to activate polymerase, 15 seconds at 95 ° C to denature the DNA strands and 1 minute at 61 ° C for hybridization and elongation from the primers. Forty cycles are performed for the last two stages.

The expression of galectins-1 and -3 is evaluated relative to the expression of a reference gene, such as that coding for β-actin, the expression of which is constant.

The probes and primers used and chosen using the Primer Express® software sold by the company Perkin Elmer are as follows: Galectin-1:

Direction: 5'-TCA ATC ATG GCC TGT GGT CTG-3 '(SEQ ID n ° 4)

Anti-sense: 5'-AAG CTC TTG GCG TCC GAG G-3 '(SEQ ID n ° 5) Probe: 5'-TCG CCA GCA ACC TGA ATC AAC CTG-3' (SEQ ID n ° 6) Galectin-3 :

Direction: 5'-AAT GGC AGA CAG CTT TTC G-3 '(SEQ ID n ° 7)

Anti-sense: 5'-GAT CAT GGC GTG GTT AGC-3 '(SEQ ID n ° 8)

Probe: 5 '-TTC CAC TTT AAC CGC CGC TTC AAT GAG AAC-3'

(SEQ ID No. 9) β-actin:

Direction: 5'-TGG CGC TTT TGA CTC AGG ATT-3 '(SEQ ID n ° 10)

Anti-sense: 5'-GGG ATG TTT GCT CCA ACC AAC-3 '(SEQ ID n ° 11)

Probe: 5'-GCC GTC GCC TTC ACC GTT CCA GTT TTT-3 '(SEQ ID n ° 12) In order to validate each microplate subjected to PCR cycles, a calibrator is prepared from the lungs of a batch of 6 mice C3H with human flora subjected to a standard diet (sterile water and RO3 granules) from which a lobe was removed. The total RNAs were extracted using the protocol previously described for the lung of the batches of mice to be tested. The different extracts thus obtained are then collected for each organ in order to constitute a pool serving as a calibrator throughout the duration of the analysis.

During the analysis, the samples to be tested are deposited in duplicate and the standard range produced from the calibrator sample is deposited in triplicate.

An average is then taken from each sample and for the calibrator sample.

The results of the relative expression of galectin-1 and -3 with respect to β-actin in the lungs of the mice are collated in Table V below: TABLE V

In Table V: - ^a = number of mice per batch;

- = median of the relative expression of galectin;

- ^c = extreme results of relative expression of galectin;

- the value designated by the letter U is the statistical value of the Mann-Whitney U test; - p indicates the significance threshold of the method.

These results show that a significant increase in the expression of galectin-1 is observed in the lungs of the mice for which the drinking water has been replaced by the filtered fraction.

On the other hand, no modification of the expression of galectin-3 is observed in the lungs of the mice for which the drinking water has been replaced by the filtered fraction.

All of these results demonstrate that the filtered fraction (immunomodulatory product obtained according to the process according to the invention) has an immunomodulatory effect and makes it possible to increase the population of Bifidobacterium and to decrease the population of Clostridium perfringens.

Claims

1. Immunomodulatory product characterized in that it is obtained according to a preparation process comprising the following steps: - seeding and incubation, under aerobic or anaerobic conditions and at a temperature between 30 and 40 ° C approximately, of Bifidobacterium comprising at least the brief Bifidobacterium strain 1-2219 in an aqueous substrate having a pH of between 6 and 8 approximately, and comprising at least the following ingredients: i) whey permeate, ii) a whey protein hydrolyzate, iii) lactose

- elimination of Bifidobacterium from the aqueous substrate;

- dehydration of the concentrated retentate;

- dissolving the dehydrated retentate in a buffer;

- column exclusion chromatography on a column having an exclusion threshold of 600 kDa from the retentate solution;

2. Immunomodulatory product according to claim 1, characterized in that the Bifidobacterium are seeded in the aqueous substrate at a rate of 1.10 ⁴ to 4.10 ⁹ colony forming units per ml of substrate.

3. Immunomodulatory product according to claim 1 or 2, characterized in that the temperature of the substrate is maintained at a value between 37 and 40 ° C throughout the duration of the incubation.

4. Immunomodulatory product according to any one of claims 1 to 3, characterized in that the pH of the aqueous substrate is maintained at a value between 6 and 8 throughout the incubation period.

5. Immunomodulatory product according to claim 4, characterized in that the pH of the aqueous substrate is maintained at a value between 6.5 and 7.5 throughout the incubation period.

6. Immunomodulatory product according to any one of the preceding claims, characterized in that the ingredients of the aqueous substrate are present in the following amounts: i) whey permeate: from 3 to 80 g, ii) whey protein hydrolyzate: from 2 to 80 g, iii) lactose: from 5 to 50 g, these amounts being given per liter of said aqueous substrate.

7. Immunomodulatory product according to claim 6, characterized in that the ingredients of the aqueous substrate are present in the following amounts: i) whey permeate: from 40 to 60 g, ii) whey protein hydrolyzate: from 5 to 15 g, iii) lactose: from 10 to 30 g, these amounts being given per liter of said aqueous substrate.

8. Immunomodulatory product according to any one of the preceding claims, characterized in that the aqueous substrate also comprises at least one additional ingredient chosen from buffer salts, yeast extracts and cysteine hydrochloride.

9. Immunomodulatory product according to claim 8, characterized in that the aqueous substrate comprises a buffer salt chosen from sodium dihydrogen phosphate and potassium dihydrogen phosphate which represents from 0.5 to 5 g per liter of aqueous substrate.

10. Immunomodulatory product according to claim 8, characterized in that the yeast extract represents from 0.5 to 5 g per liter of aqueous substrate.

11. Immunomodulatory product according to claim 8, characterized in that the cysteine hydrochloride represents from 100 to 500 mg per liter of aqueous substrate.

12. Immunomodulatory product according to any one of the preceding claims, characterized in that the elimination of Bifidobacterium of the culture medium is produced by microfiltration or by centrifugation of the aqueous substrate.

13. Immunomodulatory product according to claim 12, characterized in that the elimination of Bifidobacterium from the culture medium is carried out by centrifugation of the aqueous substrate.

14. Immunomodulatory product according to any one of the preceding claims, characterized in that the method further comprises, after the step of eliminating Bifidobacterium, an additional step of destroying the residual enzymatic activities contained in the aqueous substrate after incubation .

15. Immunomodulatory product according to any one of the preceding claims, characterized in that the exclusion chromatography is carried out on a dextran and crosslinked agarose gel.

16. Immunomodulatory product according to any one of the preceding claims, characterized in that the filtered fraction consists of a complex of polysaccharides and proteins in which the carbohydrate fraction represents from 10 to 20% by weight, the protein part representing from 80 to 90% by weight relative to the total weight of said complex.

17. Immunomodulatory product according to claim 16, characterized in that the carbohydrate fraction of the filtered fraction has the following monosaccharide composition (expressed in molar ratios relative to rhamnose): galactose: 11 to 14; mannose: 2 to 5; glucose: 3 to 6; N-acetyl galactosamine: 2.5 to 4.5; N-acetyl glucosamine 0.5 to 1.5; neuraminic acid: 4.5 to 6.5 and rhamnose: 1.

18. Immunomodulatory product according to any one of the preceding claims, as a medicament.

19. Immunomodulatory product according to any one of the preceding claims, as an immunomodulatory drug.

20. Pharmaceutical composition characterized in that it contains, as active principle, at least one immunomodulatory product obtained according to the process as defined in any one of claims 1 to 17, and at least one pharmaceutically acceptable carrier.

21. Pharmaceutical composition according to claim 20, characterized in that it is intended for oral administration and that it is in liquid or solid form.

22. Food composition characterized in that it contains, as an ingredient, at least one immunomodulatory product obtained according to the process as defined in any one of claims 1 to 17.

23. Food composition according to claim 22, characterized in that it is in the form of a milk or not preparation, fermented or not, of animal or vegetable origin, including infant formulas, for adults or for seniors.

24. Food composition according to claim 23, characterized in that it is in the form of liquid or powdered milk, fresh products, cereals, cookies (fodder), small jars, desserts, products hospital, dietetic products or nutritional supplements.