FR2764801A1 - Use of propionic bacteria for preparation of, e.g. therapeutic composition - Google Patents

Abstract

Use of propionic bacteria (PB) for the preparation of a nutritional fluid or absorbable dietetic or therapeutic composition which releases physiologically significant amounts of nitrogen monoxide (NO) in the human or animal digestive tract, is new. Also claimed are absorbable dietetic or therapeutic compositions comprising a major amount (especially > 10<9> cells/g) of PB selected for their ability to release and/or accumulate NO in an amount of \- 1 mu g/ml in YEL medium containing 550 mu M nitrate.

Description

The present invention relates to a dietary composition or

absorbable drug containing bacteria

propionics capable of releasing significant amounts of nitric oxide in the human or animal intestine.5 For many decades, it was totally ignored that nitric oxide is one of the necessary elements.

transferable to life and its maintenance; therefore, until the last four or five years, researchers did not consider the benefits associated with the presence of this oxide, either in medicine, nutrition or physiology.

It is only very recently that we have attributed

nitrogen monoxide an impressive number of phy-

siological and that it has been hypothesized that this gas could be involved primarily in such diverse functions

control of blood pressure, the cyto-

nonspecific toxicant of macrophages, platelet aggregation

silence and neurotransmission or even the control of

motor skills of the digestive tract.

Based on this supposition, the research carried out

so much on nitric oxide have multiplied and

the importance of this gas has been confirmed.

It is known that nitric oxide, which is a very unstable gas (half-life less than 5 seconds in biological systems), is produced by biosynthesis within the human or animal organism from L-arginine by a group of enzymes called NO-synthases (NOS) of which there are

two main types, namely on the one hand the constitutive NOS

ves which are expressed in particular in endothelia-

blood platelets and neurons and, on the other hand,

the inducible NOS which are expressed mainly by certain-

certain cells of the immune system (macrophages and polynu-

in particular) by vascular smooth muscle and

endothelial cells.

It should be noted that the production of NO by the inducible NOS is, by several orders of magnitude, greater than the production of NO by the constitutive NOS, but that in

in any case, this production remains relatively low.

Now, and taking into account the aforementioned beneficial role of nitrogen monoxide, it would be desirable to be able to increase

this production in particular by using the natural route of food metabolism. However, until now, means have never been proposed to achieve this result.

The object of the invention is to fill this gap.

In accordance with the invention, it has been possible to achieve the desired objective by observing that, surprisingly, a type

bacteria, propionic bacteria, are suspected

capable of producing nitric oxide, and that among these

bacteria, certain species and certain strains among these

species produce large quantities of it.

These are propionic bacteria which, although

that do not belong to the group of lactic or bi-

fidobacteria conventionally introduced into the body through dairy desserts or other fermented dairy products, have nevertheless been present in human food for centuries: it is in fact they which allow holes to be obtained during the manufacture of cheese called "Emmental" "

which at the end of ripening contains about 10 cells / g of bacteria

propionic lines.

It should be noted that the fermentation of these bacteria produces, among other things, propionic acid, acetic acid

and carbon dioxide.

The aforementioned finding is all the more surprising since it has been possible to verify that lactic acid bacteria, bifidobacteria and / or yeasts, commonly used in the food industry, do not produce monoxide.

nitrogen.

The invention relates, therefore, to the use of propionic bacteria for obtaining an absorbable dietetic or medicinal composition capable of releasing large amounts of nitrogen monoxide in

the human intestine.

In accordance with the invention, this composition may consist of a fermented liquid preparation or else

by a dehydrated preparation.

In the latter case, it is advantageously in the form of individual fractions containing the dose of bacteria to be absorbed daily. These fractions can be ingested directly or be diluted beforehand in a liquid; they can be packaged in a form that facilitates absorption: tablets, sachets of granulated powder, liquid,

It has been verified that such dehydration preparations

Concentrated concentrations of propionic bacteria stored for two years at + 4 C see their concentration drop by less than

a Log. Experience has shown that gastro-resistant capsules

aunties or not correspond to a type of partial conditioning.

particularly advantageous.

According to another characteristic of the invention,

each individual fraction contains a large number of bacteria.

ries, preferably more than 10 bacteria.

Various experiments (summarized below) made it possible to indirectly verify the very particular aptitude

propionic bacteria to produce NO from the me-

sure of the accumulation of nitrite ions NO2 by different

bacterial strains during their culture.

1 - Preliminary comparative tests Different bacterial strains (yoghurt inoculum,

bifidobacteria, lactobacillus) were cultured in pre-

sence of a reconstituted milk medium (100 ml) supplemented with

a yeast extract (10 g / l) then incubated at 38 C.

Accumulation of nitrite was measured over time. These preliminary tests were carried out under the following conditions: * incubation at 38 C for 0, 4, 7 or 10 hours, * 3 repetitions, * dosage of nitrites by Bran-Luebbe system, * estimation of the fermentation of each culture measured

by reading the absorbance at 650 nm.

Due to the nature of the extracts to be analyzed, a sample purification step was successively carried out by double centrifugation (2 x 10 min.,

4 C, 15,000 rpm), followed by ultrafiltration on cartridge

che miniprep 10 (retention of proteins of PM> 10kD) then a partial purification by passing the sample

on Waters C18 resin (55-105 gm).

This method was first tested on standard nitrite samples (Figure 1), then on Lactobacillus culture extracts incubated for 7 hours to which a known quantity was added or not.

nitrite (Figure 2).

FIG. 1 represents the colorimetric profiles obtained on a Bran Luebbe automatic analysis line: (1) of a bifidobacteria culture medium after hours of incubation, (2) of a standard solution of nitrite, (3) of this same ultrafiltered solution,

(4) of this same ultrafiltered solution passed through re-

sine C18.

FIG. 2 represents the colorimetric profiles obtained on a Bran Luebbe automatic analysis line: (1) of a Lactobacillus culture medium after hours of incubation at 38 C,

(2) (3) of a standard solution containing 410 gg of ni-

trite / l, (4) (5) of a Lactobacillus culture medium after hours of incubation at 38 C to which a known quantity of nitrite has been added in order to obtain a solution of 820 Mg / l of nitrite

(4) of this same ultrafiltered solution passed through re-

sine C18.

These samples were purified by centrifugation.

ultrafiltration and passage through C18 resin in

conditions described above.

In accordance with these tests, no accumulation of nitrite could be detected whatsoever from

inoculum of yogurt, bifidobacteria or Lactobacil-

read, regardless of the incubation time (0, 4, 7 or

hours).

2 - Measurements of the accumulation of nitrite by cultures of propionic bacteria - Analysis of the inhibition of the enzyme NO synthase

Cultures of propionic bacteria (1 g of lyo-

philisate per 100 ml of YEL medium) were placed in

tact of different concentrations of N-methyl-L-arginine

which is a known inhibitor of the enzyme NO synthase.

These tests were carried out under the conditions

following conditions: * incubation at 30 ° C for 24, 48 or 72 hours, * 5 concentrations of inhibitor, * 3 repeats for the 24 hour incubation, * stop the incubation by boiling, * purification of the product by centrifugation and passage of the extract over C18 resin,

* determination of nitrites in the medium by analysis on sys-

tème Bran-Luebbe.

First, the nitrites accumulated were measured.

mulated by propionic bacteria in the absence of inhibitor in order to establish an accumulation kinetics

nitrite as a function of the incubation time of the bacteria

ries on YEL medium.

Figure 3 shows, on the one hand, the variations in the quantity of nitrite produced (in gg / g of bacteria) as a function of the incubation time (in hours) (<) and on the other hand the variations in turbidity (absorbance at = 650 nm) also as a function of incubation time (O). This figure shows that the amount of nitrite is

maximum at 24 hours and then decreases significantly

cative after 48 and 72 hours of incubation.

We can reasonably think that this fall re-

results from the reduction of nitrite to ammonium by nitrite reductase at a time when the necessary nitrogenous substrates

to bacterial growth are less and less

dants, as evidenced by the turbidity curve.

These tests are of a nature to prove that

cultures of propionic bacteria on YEL medium allow

attempt to obtain an accumulation of nitrite in the medium.

To verify that this accumulation of nitrite re-

Following the enzymatic production of NO, similar tests were carried out for different incubation times, and adding different concentrations

of N-methyl-L-arginine inhibitor before each incubation.

Tables I, II and III show the effects of different concentrations of N-methyl-L-arginine on nitrite production and bacterial growth (turbidity estimated by absorbent at 650 nm, respectively for incubation times of 24 hours, 48 hours and 72 hours). In the case of Table I, each value is the

mean the standard deviation for the three measurements.

TABLE I

Incubation 24 hours N-methyl -2 -1 L arginine O 1.10 1.10 1 3 6 L arginine in (mM) Nitrite (NO2_) (ieg / g bact.) 62 + 2.6 61 + 2.9 69 + 7.6 59 + 2.8 63 + 3 23 (g / g bact.) 63% inhibition in% of 0 mM Turbidity <BS X = 600.84 + 0.12 0.75 + 0.01 0.81 + 0.01 0.69 + 0.01 0.79 i 0.01 0.32 (ABS X = 650nm) Km 0 ap% ua% Z6 56% ss uoT: lTqTquj o ú T (. Dq $ /. B?) Co

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The turbidity values prove that the growth

bacterial activity is not significantly affected by inhibitor intake, regardless of the concentration

implementation and incubation time.

In contrast, the effect of the inhibitor on nitrite accumulation is evident regardless of the

incubation time. However, for a concentration don-

born, this inhibition is all the stronger as the culture time is high: thus, the highest inhibition is obtained with 6 mM of N-methyl L-arginine for 24 h of culture (Table I) and only 1 mM and 100 gM for 48 h of culture (Table II) and 72 h of culture (Table III) respectively. This effect can be explained by considering from a

apart from the fact that we are in the presence of an inhibition of the

petitive (the inhibitor being a structural analogue of the

trat) and on the other hand that the substrate concentration

gradually decreases as it is used by bacteria.

In conclusion, these effects strongly suggest that the observed accumulation of nitrite results from the NO synthase activity, i.e. the production of nitric oxide by

enzymatic route.

In view of these observations, the invention is

also relates to a dietetic or medicinal composition

absorbable teuse, characterized in that it consists of a dehydrated preparation containing a large amount, preferably more than 10 cells / g, of strains of bacteria

propionics selected according to their ability to release

manage nitric oxide at a rate of at least about 50 gg / g of

bacteria.

In accordance with the invention, it has in fact been possible to prove

ver that the accumulation of NO depends on the species or strains of

propionic bacteria used.

This situation was verified by the tests summarized

més below: 3 - Comparison of nitrite accumulations in the culture medium in the case of 9 strains of 4 different species of propionic bacteria In accordance with this test, the strains P20, P23, 2408, 2410, 2500 and 2501 of the species

P.freudenreichii and the strains TL221, TL223 and TL207 ap-

leaving respectively to the species P. thoenii,

P.acidipropionici and P.jensenii.

It should be noted that the TL strains (milk technology

tière) are strains belonging to 'INRA, while strain P23 was registered at the National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute

under number 1-1804 dated 18.12.96.

The different strains of propionic bacteria (1 g of lyophilisate or 5 ml of fresh culture) were cultured on 100 ml of YEL medium according to the following conditions: * incubation at 30 ° C. for 12, 24, 36 or 48 hours, * 3 repetitions , * stopping the incubation by boiling, * purification of the product by centrifugation and passage of the extract through C18 resin,

* accumulation of nitrite in the medium, measured by ana-

lysis on the Bran-Luebbe system, * estimation of the fermentation of each culture measured

by reading the absorbance at 650 nm.

The results obtained for each strain are ras-

appear in figure 4.

This represents, in each case, the variations

The accumulation of nitrite (El) and the turbidity of the culture medium (O) as a function of the incubation time. Each value corresponds to the mean

the standard deviation of the mean for n = 3.

It should be noted that the nitrite accumulation scales are 25 times greater in the

the case of strains P23 and TL223.

These results prove that the bacterial growth

nothing, estimated from the evolution of the turbidity of the mid-

The place of culture, is close for all the strains studied, reaching approximately 2 to 2.5 OD after 48 hours of incubation, with the exception of the TL221 strain for

in which turbidity only reaches 0.6 OD after 2 days.

On the other hand, there are highly differences

significant for the accumulation of nitrite based on

tion of time.

Indeed, the strains 2500, 2408, P20, 2501, 2410,

TL207 and TL221 accumulate a relatively small quantity

load-bearing nitrite, the maximum (0.1 gg of NO2 / ml) being reached respectively after 36, 12, 36, 12, 12, 24 and

24 hour incubation.

In contrast, a much stronger accumulation of

nitrite was obtained with strains P23 and TL223 which ac-

accumulate, at most, 1.8 lg of NO2 / ml after, respectively

ment, 36 and 24 hours of incubation.

It should be noted that the strain of propioni-

ques analyzed in the second test mentioned above (Figure 3; Tables I, II and III) had an intermediate position with a maximum accumulation of NO2, of approximately

0.5 gg / ml.

These tests have therefore made it possible to observe that there are significant differences between the quantities of nitrite which can be produced by different strains of propionic bacteria from four different species, these differences being independent of the growth of these strains. These results could be confirmed by the study for each strain of the evolution of the nitrite concentration of the culture medium as a function of its turbidity and

therefore approximately bacterial growth.

The results of these latter tests are reported in Figure 5 in which each value corresponds to the

mean the standard deviation of the mean for n = 3.

In view of these results, the invention relates to

also relates to a dietetic or medicinal composition

absorbable teuse of the aforementioned type, characterized in that it contains propionic bacteria belonging to

strain P23 of the species P.freudenreichii.

The invention also relates to a composition for

absorbable drug or drug of this type, characterized by

Sée in that it contains propionic bacteria belonging to strain TL223 of the species

P. acidipropionici.

The aforementioned tests were of a nature to

It is clear that, among the strains studied, strain TL223 is the most highly accumulator of nitrite. This strain was therefore retained in the context of an additional test relating to the direct measurement of the production of nitric oxide.

4 - Measurement of the production of NO by strain TL223 under at-

helium mosphere In accordance with these tests, the cultures were carried out in 10 ml tubes containing 5 ml of medium

YEL and 0.25 ml of fresh culture of strain TL223.

The atmosphere in the tubes was immediately evacuated

by a flow of helium (100 ml / min.) for 100 seconds.

The accumulation of NO in the atmosphere of the tubes was then measured over time under the following conditions: * incubation at 30 ° C. for 24, 48 or 72 hours, * 4 repetitions,

* measurement of NO accumulation by spectrometry analysis

sort by mass, * estimate of the fermentation of each culture measured

by reading the absorbance at 650 nm.

During a preliminary test, the purification system

gas tion (Roboprep G +) - mass spectrometer (Twenty-

Twenty) was calibrated by increasing amounts of mo-

nitrogen oxide.

This gas was generated from NaNO2 in the presence

of a solution of KI and H2SO4.

The identification and quantification of nitrogen monoxide 14 16 were carried out by its mass: 30 for N 0 and

16 14 17

31 for N O and N O. This identification was then confirmed by measuring the isotope ratio:

16 14 17 14 16

31/30 = [1 N O60 + N O] / N O. It should be noted that the ratio

The theoretical 31/30 isotopic port of NO is 0.00367 in the absence of 1.7 o contamination.

The results of this preliminary test are reported

shown in Figure 6 in which the left part (A) corresponds to the calibration curve of the mass spectrometer used for the quantification of nitrogen monoxide while the right part (B) corresponds to the measurement of

isotope ratio 31/30.

The actual results of this test are

reported in figure 7.

More precisely, FIG. 7A represents the va-

riations of the accumulation of NO in the atmosphere of the tubes

bes as a function of the turbidity of the medium while FIG. 7B represents the variations of this accumulation

depending on the incubation time.

Vertical or horizontal bars indicate, when wider than the symbol, the standard deviation

of the mean for n = 4.

A comparison of FIG. 7B (which represents the evolution over time of the turbidity of the culture medium under helium) with FIG. 4 (which represents this

same evolution in air) shows that the growth of the

che TL223 is not significantly affected by an at-

mosphere consisting mainly of helium.

It has also been established that the rate of accumulation of nitrogen monoxide in the atmosphere is constant during about the first 45 hours of incubation, then decreases thereafter (Figure 7B), which

corresponds to a turbidity close to 1.5 OD (figure 7A).

After about 65 hours of incubation (high turbidity

less than 1.7 OD), about 1.5; g of NO are accumulated

in the helium atmosphere for 1 ml of the culture medium.

It can be assumed that this figure and the NO dosages could be slightly underestimated due to a weak but constant contamination by oxygen of the air during the analysis by mass spectrometry (oxidation of the

nitrogen monoxide to nitrite).

The order of magnitude obtained is nevertheless compatible with the nitrite contents measured in the medium for cultures in contact with air (test No. 2). In the latter case, it was in fact observed (Figure 5) that the TL223 strain accumulated a maximum of 1.8 mg of NO2 / ml for a turbidity of 1.5 OD, which corresponds to

at about 1.2 µg NO / ml.

These results seem to prove that the procedure used (helium atmosphere) does not completely block the

transformation of NO into nitrite within the medium. We have-

could in fact have expected that the quantity of NO found in the helium atmosphere (cumulative phenomenon)

is very much greater than the quantity of nitrite ac-

cumulated in the medium (transient phenomenon).

Nevertheless, the values obtained for the clearance

* nitrogen monoxide in an aerobic atmosphere must be

flabbergasted as significant.

It should be noted that, in accordance with another character

teristic of the invention, it may be advantageous to add an arginine supplement or a product rich in

arginine in the composition in accordance with the invention.

According to another characteristic of the invention, this composition can also contain other bacteria

such as bifidobacteria and / or lactic acid bacteria.

The benefits associated with the ingestion of propionic bacteria have, moreover, been verified by investigations

carried out in vivo in healthy humans.

- Study of the effect of the ingestion of propionic bacteria on intestinal transit in healthy humans This study was carried out in a hospital environment in

CHU de Caen on a series of 19 male subjects voluntarily

healthy res.

At the start of this test, we absorbed daily-

nment for each volunteer 10 radiopaque markers, this

for 8 consecutive days, in accordance with the protocol

written in Arhan P, Devroede G, Jehannin B et al. Dis Colon Rectum 1981; 24: 625-9. and Bouchoucha M, Devroede G. Arhan P et al. Dis Colon Rectum 1992;

: 773-82.

According to this protocol, the study of the transit is carried out by counting the radiopaque markers ingested in the different areas of the abdominal cavity distributed over a frontal abdomen image. These areas (right colon, colon

left and rectosigmoid) are defined by fic-

ieme tives joining the 5 lumbar vertebra to the contour of the pelvic cavity. Transit time is calculated using the formula T = 1 / N. not. At; N being equal to 10 markers, n representing the number of markers counted in a region

and At being equal to 24 hours.

The day following this ingestion, that is to say the ith

9th day, the volunteers were subjected to an X-ray

front abdomen without preparation.

From the next day, that is to say from the i me

day, we ingested every day

voluntarily, for 2 weeks, a capsule containing 10 propionic bacteria from a food bank

ches used in the cheese industry, therefore perfect-

ment harmless to humans.

A second transit time study similar to the first was performed during the second week of propionics ingestion, i.e. from 17i to

26th day.

This study revealed a significant slowing of the transit time of the left colon (p <0.05 according to the Wilcoxon Matched-Paired Signed-Ranks statistical test

Test.); transit times of the right colon and recto

sigmoid were not significantly altered by

ingestion of propionics.

This study therefore made it possible to prove that the ingen-

tion of propionic bacteria has an influence on the mo-

intestinal tricity; we can assume that these results

are linked to the synthesis of nitric oxide by bacteria

propionic lines.

R E V E ND I C A T IONS

1) Use of propionic bacteria for obtaining a dietetic or medicinal composition which can be absorbed with suspicion.

capable of releasing significant amounts of nitric oxide5 in the human intestine.

2) Use according to claim 1, characterized in that

the absorbable dietary or drug composition consists of a dehydrated preparation.

3) Use according to claim 2, characterized in that

the composition is in the form of individual fractions

containing them the dose of bacteria to be absorbed daily

daily. 4) Use according to claim 3, characterized in that

each individual fraction contains more than 10 bacteria.

) Use according to claim 1, characterized in that the absorbable dietetic or medicinal composition is

consisting of a fermented liquid preparation.

6) Absorbable dietetic or medicinal composition, characterized in that it consists of a dehydrated preparation containing a large amount, preferably more than 10 cells / g of strains of propionic bacteria selected according to their ability to release nitrogen monoxide at least

at least 50 gg / g of bacteria.

7) Composition according to claim 6, characterized in that

it contains propionic bacteria belonging to the

che TL223 of the species P.acidipropionici.

8) Composition according to claim 6, characterized in that it contains propionic bacteria belonging to the strain P23 of the species P. freudenreichii.5.

9) Composition according to any one of claims 6 to

8, characterized in that

it further contains an arginine supplement or an arginine-rich product.

) Composition according to any one of claims 6 to

9, characterized in that

it also contains other bacteria such as bifi-

dobacteria and / or lactic acid bacteria