Patent Application
20050036996
The present invention relates to an absorbable common food composition or an absorbable dietary or medicinal composition containing propionibacteria which are capable of releasing physiologically significant amounts of nitric oxide into the human or animal digestive tract.
For many decades, it has been entirely ignored that nitric oxide is one of the elements necessary for life and for maintaining life; consequently, up until 4 or 5 years ago, researchers paid little attention to the benefits associated with the presence of this oxide, either in medicine, in nutrition or in physiology.
It is only very recently that an impressive number of physiological functions have been attributed to nitric oxide and that the hypothesis was put forward that this gas might be involved extensively in functions as diverse as controlling arterial pressure, the non-specific cytotoxic function of macrophages, platelet aggregation and neurotransmission, or controlling the motility of the digestive tract.
Starting with this assumption, research related to nitric oxide mushroomed and the importance of this gas was able to be confirmed.
It is known that nitric oxide, which is a very unstable gas (half-life of less than 5 seconds in biological systems), is produced by biosynthesis in the human or animal body from L-arginine by a group of enzymes known as the NO-synthases (NOS), of which two main types exist, namely, on the one hand, the constituent NOSs which are expressed in particular in the endothelial cells, the blood platelets and the neurons, and, on the other hand, the inducible NOSs which are expressed mainly by certain cells of the immune system (macrophages and polymorphonuclear leukocytes in particular), by vascular smooth muscle and endothelial cells.
It should be noted that the production of NO by the inducible NOSs is greater than the production of NO by the constituent NOSs by several orders of magnitude, but that, in any case, this production remains relatively small.
Now, given the abovementioned beneficial role of nitric oxide, it would be desirable to be able to increase this production in particular by using the natural route of food metabolism.
However, no means for achieving this result have ever been proposed hitherto.
The object of the invention is to fill in this absence.
In accordance with the invention, it has been possible to achieve the desired aim by observing that, surprisingly, bacteria of one specific type, the propionibacteria, are capable of producing nitric oxide, and that, among these bacteria, certain species and certain strains among these species produce it in large amounts.
They are propionibacteria which, although not belonging to the group of lactic bacteria or bifidobacteria conventionally introduced into the body via milk-based desserts or other fermented dairy products, have nevertheless been present in human food for centuries: indeed, it is these bacteria which produce the holes during the manufacture of the cheese known as “emmental” which, after maturing, contains about 109 cells/g of propionibacteria.
It should be noted that the fermentation of these bacteria produces, inter alia, propionic acid, acetic acid and carbon dioxide.
The abovementioned finding is all the more surprising since it has been possible to confirm that lactic acid bacteria, bifidobacteria and/or yeasts, commonly used in the agrifood sector, do not produce carbon monoxide.
The invention consequently relates to the use of propionibacteria to produce an absorbable common food composition or an absorbable dietary or medicinal composition which is capable of releasing physiologically significant amounts of nitric oxide into the human or animal digestive tract.
In accordance with the invention, this composition can consist of an elaborate preparation and/or can be presented in liquid form (in particular a fermented liquid), in dehydrated form or in a form of intermediate moisture content.
More specifically, it should be noted that, without, however, departing from the scope of the invention, the composition can be:
In accordance with the invention, the propionibacteria can be introduced in the form of a biomass or in the form of a leaven capable of multiplying in situ.
When it is dehydrated, the composition is advantageously in the form of individual fractions containing the dose of bacteria which needs to be regularly absorbed.
These fractions can be ingested directly or prediluted in a liquid; they can be packaged in a form which facilitates absorption: tablets, sachets of granulated powder, liquid, etc.
It has been confirmed that such concentrated dehydrated preparations of propionibacteria stored for one year at ±4° C. undergo a fall in concentration of less than one Log unit.
Experience has shown that gelatin capsules, which may or may not be gastroresistant, are a particularly advantageous type of packaging.
According to another characteristic of the invention, each individual fraction contains a large number of bacteria, preferably more than 109 bacteria.
Various experiments (summarized below) confirmed the quite specific ability of different strains of propionibacteria to produce NO during their culturing, firstly indirectly by measuring the nitrite ions NO2 [sic], and then directly by mass spectrometry analysis in anaerobic medium.
During these experiments, the nitrite concentration in arginine-rich and nitrate-poor (50 μM) media was studied, in a first stage, and it was realized that arginine is not a determining factor in the observed production of NO.
In a second stage, nitrate-supplemented media were investigated. The results obtained in the latter case revealed the nitrate-dependent nature of the production of nitric oxide.
1—Comparative Preliminary Tests
Various bacterial strains (yoghurt inoculum, bifidobacteria, lactobacillus) were cultured in the presence of a reconstituted milk medium (100 ml) supplemented with a yeast extract (10 g/l) and then incubated at 37° C.
The accumulation of nitrite was measured over time.
These preliminary tests were carried out under the following conditions:
On account of the nature of the extracts to be analyzed, a step of purification of the samples was subsequently carried out by a double centrifugation (2×10 min., 4° C., 15,000 rpm), followed by an ultrafiltration on a Miniprep 10 cartridge (retention of the proteins of MW>10 kD) and then partial purification by passing the sample through Waters C18 resin (55-105 μm).
This method was tested, in a first stage, on standard nitrite samples (
These samples were purified by centrifugation-ultrafiltration and passage through C18 resin under the conditions described above.
In accordance with these tests, no accumulation of nitrite could be detected, either using yoghurt inoculum, bifidobacteria or Lactobacillus, irrespective of the incubation time (0, 4, 7 or 10 hours).
2—Demonstration of the Accumulation of Nitrite by Propionibacterium Cultures
The possible presence of nitrate or nitrite in the preparation of the YEL medium was investigated beforehand by calorimetric assay (Boehringer kit): it was thus possible to demonstrate the presence of an appreciable amount of nitrate in this medium (concentration of about 50 to 100 μM) which might come from the yeast extract used for the manufacture of this medium; on the other hand, it was confirmed that the YEL medium was totally free of nitrite.
Propionibacterium cultures (1 g of lyophilisate per 100 ml of YEL medium) were tested.
These tests were carried out under the following conditions:
The nitrites accumulated by the propionibacteria were assayed in order to establish kinetics of nitrite accumulation as a function of the incubation time of the bacteria on YEL medium.
This figure shows that the amount of nitrite is at a maximum at 24 hours and then decreases significantly after 48 and 72 hours of incubation.
It may reasonably be considered that this fall results from the reduction of the nitrite to NO, N2O or other compounds by nitrite reductase.
In accordance with the invention, it was possible to prove that the accumulation of NO2 depends on the propionibacterium species or strains used.
This situation was confirmed by the tests summarized below:
3—Demonstration and Comparison of the Nitrite Accumulations in the Culture Medium in the Case of 9 Strains of 4 Different Propionibacterium Species
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 belonging, respectively, to the species P. thoenii, P. acidipropionici and P. jensenii, were studied.
It should be noted that the TL (technologie laitiére [dairy technology]) strains are strains belonging to the INRA-LRTL, while the strain P23 (or ITG23) was registered at the Collection Nationale des Cultures de Micro-organismes [National Collection of Microorganism Cultures] (CNCM) of the Institut Pasteur under the number I-1804 dated 18.12.96.
The various propionibacterium strains (1 g of lyophilisate or 5 ml of fresh culture) were cultured on 100 ml of YEL medium containing about 50 μm of nitrate, under the following conditions:
The results obtained for each strain are collated in
This represents, in each case, the variations in the accumulation of nitrite (□) and in the turbidity of the culture medium (◯) as a function of the incubation time. Each value corresponds to the average±the standard error of mean for n=3.
It should be noted that the scales of nitrite accumulation are 25 times greater in the case of the strains P23 and TL223.
These results prove that the bacterial growth, estimated from the change in turbidity of the culture medium, is similar for all of the strains studied, reaching about 2 to 2.5 OD after incubation for 48 hours, except for the strain TL221 for which the turbidity reaches only 0.6 OD after 2 days.
On the other hand, highly significant differences exist as regards the accumulation of nitrite as a function of time.
Specifically, the strains 2500, 2408, P20, 2501, 2410, TL207 and TL221 accumulate a relatively small amounts of nitrite, the maximum (0.1 μg of NO2−/ml) being reached after incubation for 36, 12, 36, 12, 12, 24 and 24 hours of incubation, respectively.
In contrast, a much greater accumulation of nitrite was obtained with strains P23 and TL223, which accumulate, at the maximum, 1.8 μg of NO2−/ml after 36 and 24 hours of incubation, respectively.
It should be noted that the propionibacterium strain analyzed in the abovementioned second test (
These tests thus made it possible to state that significant differences exist between the amounts of nitrite which can be produced by different propionibacterium strains from four different species, these differences being independent of the growth of these strains.
These results could be confirmed by studying the change in the nitrite concentration of the culture medium as a function of its turbidity and thus, approximately, of the bacterial growth, for each strain.
The results of these last tests are given in
The abovementioned tests were designed to establish that, among the strains studied, the strain TL223 accumulates nitrites to the greatest extent, these nitrites disappearing after 12 hours. This strain was thus selected in the context of complementary tests relating to the direct measurement of the production of nitric oxide by mass spectrometry analysis in anaerobic medium.
4—Preliminary Measurement of the Production of NO by the Strain TL223 Under a Helium Atmosphere
In accordance with these tests, the cultures were prepared in 10 ml tubes containing 5 ml of YEL medium containing about 50 μM of nitrate and 0.25 ml of fresh culture of the strain TL223.
The atmosphere of 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:
During a preliminary test, the gas purification system (Roboprep G+)—mass spectrometer (Twenty-Twenty) was calibrated with increasing amounts of nitric oxide.
This gas was generated from NaNO2 in the presence of a solution of KI and H2SO4.
The identification and quantification of the nitric oxide were performed on the basis of its mass: 30 for 14N16O and 31 for 15N16O and 14N7O. This identification was then confirmed by measuring the isotope ratio: 31/30=[15N16O+14N17O]/14N16O. It should be noted that the theoretical isotope ratio 31/30 for NO is 0.00367 in the absence of contamination with 17O.
The results of this preliminary test are given in
The actual results of this test are given in
More specifically,
The vertical or horizontal bars indicate, when they are wider than the symbol, the standard error of mean for n=4.
A comparison of
It was also possible to establish that the rate of accumulation of nitric oxide in the atmosphere is constant over about the first 45 hours of incubation, and then curves off (
After incubation for about 65 hours (turbidity greater than 1.7 OD), about 1.5 μg of NO are accumulated in the helium atmosphere per 1 ml of culture medium.
The order of magnitude obtained is compatible with the nitrite contents measured in the medium of cultures in contact with air.
In the latter case, it was in fact found (
From this preliminary direct measurement of the production of NO by the strain TL223, an attempt was made, in accordance with the invention, to confirm the route of synthesis of this nitric oxide, and the notion of investigating whether or not this synthesis is stimulated by a supply of nitrite or nitrate was proposed for this purpose.
Such a stimulation was able to be confirmed by means of the tests summarized below:
5—Study of the Stimulation of the Production of NO by Supplying Nitrite or Nitrate
With the aim of investigating whether or not the production of NO by propionibacteria is possible from NO2− or NO3−, an investigation to see whether or not an increase in the production of NO by the strain TL223 when it is in the presence of 1 mM KNO2 or KNO3 (with and without isotopic labeling with 15N) is observed was carried out.
This experiment was carried out under the following conditions:
The accumulation of NO as a function of the NO3− concentration (100, 150, 350, 650 and 1050 μM) or of the NO2− concentration (50, 100, 400, 800, 1000 μM) after incubation for 72 hours at 30° C. (TL223 inoculated at a concentration of 1% in a YEL medium) was also analyzed.
The samples to be analyzed were distributed in NO analysis tubes whose atmosphere was immediately evacuated by flushing with helium for 150 s in order to obtain strict anaerobic conditions.
The effect of flushing with helium on a possible detection of NO by the mass spectrometer was tested on a sterile YEL medium: this YEL medium, preflushed with helium, was incubated at 30° C. for 72 hours. After 0, 24, 48 and 72 hours of incubation (3 repetitions), no production of NO was detected and the turbidity values remained at 0.
It was thus possible to check the quality of the helium flush, the absence of any bacterial contamination and the absence of interaction between the helium flush and the measurement of NO by mass spectrometry.
The abovementioned tests gave the results reported in
More specifically:
Each of these figures relates to the strain TL223 cultured on a YEL medium alone, in the presence of 1 mM nitrate (15NO3−—labeled to a proportion of 50% and 14NO3−) and in the presence of 1 mM nitrite.
The vertical bars represent the ±standard error of mean for n=3 when they are larger than the symbol.
These figures prove that the accumulation of NO by TL223 cultured on 1 mM KNO3 (labeled with nitrogen-15 or unlabeled) or on 1 mM KNO2 is close to 7 μg of NO/ml after incubation for 48 hours at 30° C.; this value is 3.5 times greater than the production of NO in the case of a YEL medium not supplemented with nitrate or nitrite (
It was also observed that when the strain TL223 is cultured on K15NO3, the profile of the peak of mass 31 (15N16O) increases to a great extent relative to the NO analyzed on a YEL medium containing unlabeled nitrate, thereby confirming this situation.
Furthermore, the addition of 1 mM unlabeled KNO3 or KNO2 leads to a production of NO with an isotope ratio of 0.75% (incubation for 48 hours—
These last observations very clearly confirm that the gas analyzed by mass spectrometry was indeed NO.
These tests thus made it possible to state that the strain TL223 is capable of synthesizing NO directly from nitrite or in the presence of nitrate after reducing this nitrate to nitrite.
On the basis of the isotope ratio values obtained on YEL medium supplemented with 1 mM K15NO3 labeled to a proportion of 50%, it is possible to deduce the degree of conversion of nitrate into nitrite: thus, about 20% of the K15NO3 supplied is converted into nitric oxide by the strain TL223.
The nitrate, initially present in the sterile YEL medium, explains the observed NO production by TL223 (of about 2 μg of NO/ml after incubation for 72 hours—
The variations in NO production as a function of the nitrite or nitrate concentration are represented in
In these figures, the nitrate concentrations have been corrected taking into account the presence of about 50 μM nitrate in the YEL medium alone, and are as follows: 100, 150, 350, 550, 650 and 1050 μM.
The nitrite concentrations are as follows: 50, 100, 400, 800 and 1000 μM.
These figures show that, for the ranges chosen, the production of nitric oxide by the strain TL223 is proportional to the initial concentration of nitrate (
In both cases, no plateau phase was observed, which leads to the assumption that the nitrate or nitrite concentrations used do not make it possible to obtain the maximum level of accumulation of NO by the propionibacterium TL223.
It should also be noted that the presence of high concentrations of nitrate or nitrite do not affect the bacterial growth, given that the turbidity values at 72 hours are very similar for all the concentrations which were tested.
It should moreover be pointed out that the curves obtained in
Furthermore, these results show that, with the strain TL223, the step of reduction of nitrate to nitrite is not limiting for the nitrate concentrations chosen in this experiment.
It is also important to point out that the degree of conversion of the NO3— (
This suggests that the production of NO is strongly regulated and that it is predominant over the use of nitric nitrogen for the nitrogenous syntheses.
From the abovementioned conclusions, the production of NO was studied in 12 strains of propionibacteria of two different species. The results of these tests are summarized below:
6—Study of the Production of NO by Different Propionibacterium Strains
In accordance with this test, the propionibacterium strains selected were the following:
It should be noted that the strains CNRZ80, CNRZ81, CNRZ86, CNRZ89, CNRZ277 and CNRZ287 belong to the INRA-CNRZ public collection, while the strain NCDO1072 belongs to the British collection “National Collection of Dairy Organisms”; the other strains belong to private collections.
After inoculation on YEL medium in the presence of 550 μM nitrate, the bacterial cultures were immediately distributed in 5 ml units in sealed tubes and flushed with helium (anaerobic conditions). Next, the propionibacterium strains were subjected to the following experimental conditions:
The vertical bars represent the ±standard error of mean for n=3 when they are larger than the symbol.
This figure shows the existence of large divergence between the propionibacterium strains.
Globally, by comparing the levels of production of NO after incubation for 72 hours, the strains can be classified in three categories:
It should be noted that, in the strains belonging to the first two categories, the NO produced does not decrease at the end of growth and that it therefore does not appear to be reused by the propionibacteria: there is thus accumulation of NO.
The vertical bars represent the +standard error of mean for n=3, when they are larger than the symbol.
The strain TL223 is represented in each case for comparative purposes.
The vertical bars represent the standard error of mean for n=3, when they are larger than the symbol.
The strains which produce the most NO (TL223, CNRZ80, NCDO1072 and PR75) all reach high turbidity values (4 to 5 OD units after incubation for 72 hours).
Among these strains, it should be noted that the bacteria NCDO1072 and PR75 grow less quickly than TL223, but are capable of accumulating large concentrations of NO very rapidly.
Thus, PR75 produces 2.8 μg of NO/ml for a turbidity of 0.5 OD units. The maximum NO production is recorded for turbidity values of about 1.5 OD units for NCDO1072 and PR75, compared with 3.4 OD units for TL223.
In order to “refine” these results, the change in the NO, nitrate and nitrite concentrations of the culture media for the 13 strains of propionibacterium studied, as a function of the incubation time was analyzed.
The results obtained are collated in the table below, in which the concentrations are expressed in μM. For each strain, the measurements were taken on a tube, after removal of the bacteria by centrifugation.
This table allows the following observations to be made:
The tests summarized above revealed that certain propionibacterium strains are capable of reducing the nitrate in the culture medium and thus producing the nitrite required for the synthesis of NO.
It should be noted that the propionibacterium strains which produce the most NO (TL223, CNRZ80, NCDO1072 and PR75) all belong to the species P. acidipropionici and, moreover, all have nitrate reductase activity.
In contrast, the strains apparently producing less NO, or even none at all (detection limit of the mass spectrometer) are also bacteria which have no known nitrate reductase activity (LS410, LS2501 and CNRZ287).
For certain propionibacterium strains, it appears that the NO production kinetics are not directly linked to the nitrate reductase activity.
Given these results, the invention also relates to an absorbable dietary or medicinal composition, characterized in that it consists of a preparation containing a large amount, preferably more than 109 cells/g of propionibacterium strains selected as a function of their capacity to release and/or accumulate nitric oxide in a proportion of at least 1 μg/ml of YEL medium containing 550 μM nitrate.
According to another characteristic of the invention, this composition contains propionibacteria belonging to at least one of the strains TL223, CNRZ80, CNRZ86 and NCDO1072 of the species P. acidipropionici.
Among these strains, the strain TL223 proved to be particularly advantageous.
It should also be noted that the strain CNRZ80 is of quite specific value from a point of view of “productivity” given that it is capable of accumulating a high concentration of NO, and of doing so very rapidly (for relatively low turbidity values).
According to another characteristic of the invention, this composition contains propionibacteria belonging to at least one of the strains ITG23, CNRZ81, CNRZ89, CNRZ2.77 and LS2502 of the species P. freudenreichii.
It should be noted that, in accordance with another characteristic of the invention, the composition can also contain other bacteria, such as bifidobacteria and or lactic acid bacteria.
In order to complete the results obtained above, tests were carried out on two types of non-propionic bacteria: E. coli and Lactobacillus farciminis, known for their ability to reduce nitrites.
The results of these tests are summarized below:
7—Study of the Production of NO by the Strains E. coli and L. farciminis
These complementary tests were mainly carried out given the existence of the publication “Heme-dependent and heme-independent nitrite reduction by lactic acid bacteria results in different N-containing products” Gudrun Wolf, Elke K. Arendt, Ute Pfähler and Walter P. Hammes—International Journal of Food Microbiology, 10 (1990) 323-330) which mentions that certain lactic acid bacteria (L. farciminis) are capable of producing nitric oxide from nitrite.
Preliminary experiments showed that after growing the strain L. farciminis for 5 h 30 min in MRS supplemented with 1 mM nitrate, nitrate and nitrite were no longer detected in the culture medium.
The same observation was made as regards the strain E. coli after growth for 7 h 30 min on BHI medium supplemented with 1 mM nitrate.
It is known that, during its growth, the strain L. farciminis acidifies the MRS medium (about pH 5 after culturing for 5-6 hours).
It was possible to observe, from tests carried out on YEL medium, that nitrites are converted into NO in acidic medium.
These tests were carried out under the following experimental conditions:
The results obtained are collated in
This figure is able to prove that there is appreciable production of NO from the nitrite in the medium when this medium is acidic, this production increasing as the pH decreases.
Consequently, comparative tests of NO production by L. farciminis and by E. coli which is reputed not to produce NO (Brittain T, Blackmore R, Greenwood C & Thomson A J (1992)—Bacterial nitrite—reducing enzymes—Eur. J. Bio. Chem., 209, 793-802), were carried out.
These tests were carried out under the following conditions:
These tests gave the results reported in
More specifically:
It should be noted that the values obtained for the NO production are all markedly lower than the threshold of 1 μg/ml which was considered above as significant: the result of this is that there is no accumulation of NO in the culture tubes.
These results thus indicate that the absence of nitrate and nitrite observed in the preliminary experiment after 5 h 30 min (L. farciminis) and 7 h 30 min (E. coli), respectively, is not compensated for by an accumulation of NO which might be either of chemical origin (associated with acidification of the medium) or of bacterial origin.
In the case of the L. farciminis strain, these results were confirmed by tests carried out on bacteria in the form of resting cells at a pH adjusted to 6.5 by a phosphate buffer containing lactate, under the following operating conditions:
This analysis gave the results reported in
These results confirm those obtained above, i.e. that the amounts of NO produced are too small to be significant and thus that the L. farciminis strain is not capable of accumulating nitric oxide. However, it is possible that this strain produces nitric oxide at the start of growth, but that any NO produced is reused by the bacterium.
Complementary tests were carried out on the strains TL223 and CNRZ80 in the form of resting cells after incubation at 30 and also at 37° C.
8—Change in NO Production by Propionibacteria in the form of Resting Cells
This experiment was carried out under the following conditions:
It should be noted that, during the tests relating to the incubation at 30° C., in addition to the strains TL223 and CNRZ80, the strain CNRZ81 was examined with a twofold bacterial concentration.
This experiment gave the results reported in
These results are able to prove that there is a consequent production of NO by resting cells not only in the case of the two strains of the species P. acidipropionici (TL223 and CNRZ80), but also in the case of the strain of the species P. freudenreichii (CNRZ81). The strain TL223 is the most productive.
Globally, for identical bacterial concentrations, the NO production by resting cells is of the same order as that observed in the case of bacteria cultured on YEL medium.
The production of NO by resting cells occurs essentially during the first five hours of incubation; beyond this period, the production is low.
It was thus possible to observe that, at 37° C., the production of NO is identical (TL223) or slightly higher (CNRZ80) than that obtained at 30° C.
The advantages associated with the ingestion of propionibacteria were, in addition, confirmed by investigations performed in vivo on healthy humans.
9—Study of the Effect of Ingestion of Propionibacteria on Intestinal Transit in Healthy Humans
This study was carried out in a hospital environment at the University Hospital of Caen on a series of 19 healthy male volunteers.
At the start of this test, each volunteer was given 10 radio-opaque markers to absorb, for 8 consecutive days, in accordance with the procedure described in the publications 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; 35:773-82.
According to this procedure, study of the transit is carried out by counting the radio-opaque markers ingested in the different areas of the abdominal cavity, which are distributed on an anterior abdominal image. These areas (right colon, left colon and rectosigmoid) are defined by imaginary lines joining the 5th lumbar vertebra to the contour of the pelvic cavity. The transit time is calculated according to the formula. T=1/N n−Δt; 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 after this ingestion, i.e. the 9th day, the volunteers were made to undergo a radiography of the anterior abdomen without preparation.
Starting from the following day, i.e. the 10th day, each volunteer was given a gelatin capsule to ingest daily for 2 weeks, this capsule containing 5×1010 propionibacteria obtained from a bank of strains used in the cheesemaking industry, and thus entirely harmless to man.
A second study of the transit time similar to the first study was carried out during the second week of ingestion of the propionibacteria, i.e. from the 17th to the 26th day.
This study revealed a significant deceleration in the transit time of the left colon (p<0.05 in accordance with the Wilcoxon Matched-Paired Signed-Ranks statistical test); the transit times of the right colon and of the rectosigmoid were not significantly modified by the ingestion of propionibacteria.
This study thus proved that the ingestion of propionibacteria has an influence on intestinal motility; it can be assumed that these results are associated with the synthesis of nitric oxide by the propionibacteria.
| Number | Date | Country | Kind |
|---|---|---|---|
| 97/00885 | Jan 1997 | FR | national |
| 96/15977 | Dec 1996 | FR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09331554 | Aug 1999 | US |
| Child | 10946156 | Sep 2004 | US |
