Patent Application
20240034984
The present invention relates to a bacterial culture medium, to a method for manufacturing the culture medium, as well as to a method for culturing a bacterium, particularly effective for cultures of bacteria that are oxygen-sensitive, even very much so, or strict anaerobes. The culture medium of the invention makes it possible for example to culture intestinal anaerobic bacteria, for example Faecalibacterium prausnitzii (F. prausnitzii). The culture medium of the present invention makes it possible in particular to manufacture the new generations of probiotics, in particular those intended to prevent and treat chronic inflammatory diseases of the intestine in strict accordance with legislation on human or animal food.
In the text below, the term “Reference” followed by a digit refers to the list of the bibliographic references presented after the “Examples” section below.
Originally, probiotics (“PBX”) were microorganisms, bacteria or yeasts, which, ingested in adequate quantities and potentially being integrated into certain food products for humans or animals, are supposed to confer a health benefit. The concept of probiotics was then extended to treatments for treating or preventing various conditions. It has indeed been demonstrated that probiotics have numerous advantages for human health and that they play a key role in normal digestive processes and in the maintenance of human or animal health. Agricultural applications of probiotics, in particular animal, fish, and plant production have gradually increased. However, there are a certain number of uncertainties concerning the technological, microbiological and regulatory aspects.
As an example, mention may be made of F. prausnitzii, the most abundant bacterium in the intestinal microbiota. It represents from 3.5 to 5% of the commensal bacteria. This bacterium is one of the major producers of butyrate, a short chain fatty acid, which is involved in gastrointestinal operations, and has numerous beneficial effects such as the defense of the intestinal mucosa, a motor effect on colon motility, a trophic effect on intestinal and colic epithelium, an effect on the defenses of intestinal mucosa by regulation of the secretion of mucus in the colon, making it possible to maintain a constant pH in the vicinity of epithelial cells, an effect on colorectal neoplastic cells, etc. (reference 1). However, this bacterium has extreme oxygen sensitivity, which makes it tricky to grow and handle. The need to grow it in an anaerobic chamber makes it technically difficult to produce and requires great expertise (reference 1). Furthermore, legislation in terms of human food is very strict, which further limits the current culture capacity of this microorganism, and more generally microorganisms having such oxygen sensitivities. Indeed, in particular, the culture medium must be devoid of compounds of animal origin.
The culture medium which was the most used until recently is the LYHBHI medium described in the document Sokol, H. et al. (2008), F. prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients, Proc. Natl. Acad. Sei. U. S. A. 105, 16731-16736 (reference 2). However, this culture medium comprises compounds of animal origin and therefore cannot be used to produce probiotics in accordance with human dietary requirements.
There is therefore a real need for a culture medium that makes it possible both to culture bacteria that are sensitive or even extremely sensitive to oxygen or strict anaerobes, and to produce probiotics in accordance with strict human or animal dietary requirements.
The aim of the present invention is precisely to meet these needs by providing a bacterial culture medium comprising, per liter of culture medium, the following components:
According to the invention, the alkali metal acetate can be chosen from a sodium or potassium acetate, preferably anhydrous, for example sodium acetate or anhydrous potassium.
According to the invention, the yeast extract may for example be autolytic yeast extract, for example a lyophilized extract, for example resulting from the autolysis of yeast cells of Saccharomyces cerevisiae, grown on molasses, for example beetroot, soy or sugarcane. It may for example be a yeast extract with CAS number 8013-01-2.
According to the invention, the maltose monohydrate is preferably D-(+)-maltose monohydrate with CAS number 6363-53-7. The maltose is the source of sugar in the culture medium. Alternatively or in a mixture, cellobiose or other disaccharides, or the monosaccharides that compose them, may also be used.
According to the invention, the soluble salt of L-cysteine is preferably L-cysteine monohydrated hydrochloride. This component is a reducing component, which makes it possible to capture the molecular oxygen and to maintain a low redox potential in the culture medium.
According to the invention, the pantothenic acid may for example be the salt D-calcium pantothenate, the active form of vitamin B5.
According to the invention, the pyridoxal or the salt thereof may for example be pyridoxal hydrochloride or pyridoxal phosphate. This component can be replaced by pyridoxamine and pyridoxine.
The inventors found that this culture medium of the invention, which they have defined during their numerous experiments and work, with the particular trio of biotin, pantothenic acid or a salt thereof and pyridoxal, is particularly effective for cultures of bacteria that are oxygen-sensitive, even very much so, or strict anaerobes, even though it does not comprise any compounds of animal origin. The culture medium of the present invention provides excellent bacterial culture and growth performance, identical or even superior to culture media of the prior art comprising compounds of animal origin.
Furthermore, the culture medium of the present invention has numerous other advantages recorded by the inventors during the experiments, in particular: it does not foam, and therefore does not require the addition of anti-foaming agents; it does not precipitate, contrary to certain media of the prior art; and the number of components necessary for a quality bacterial culture is reduced, with only eight components.
Furthermore, the experiments carried out show that the bacterial performance in terms of production of probiotic compounds is preserved, or even improved. For example, in the case of cultures carried out with F. prausnitzii on the culture medium of the present invention, the measurements show that the production of butyrate in the medium is similar or even greater than that produced in media comprising compounds of animal origin, for example in comparison with a brain-heart infusion culture medium.
The culture medium of the present invention therefore advantageously makes it possible to produce new generations of probiotics from anaerobic bacteria, including extremely oxygen-sensitive ones or strict anaerobes, including but not limited to F. prausnitzii, that are intended to prevent and treat chronic inflammatory bowel diseases, in strict accordance with legislation on human or animal food.
The culture medium of the present invention can advantageously be used for culturing anaerobic, microaerophilic or strict, Gram-negative or positive anaerobic bacteria, for example for laboratory study purposes, seeking growth promoting treatments or intended to combat these bacteria, for example in food and/or in the field of medicine. Examples of bacteria that can advantageously be cultivated on the medium of the present invention include intestinal anaerobic bacteria, for example F. prausnitzii, Blautia, for example Blautia hydrogenotrophica and Blautia obeum.
The present invention also relates to a method for producing a culture medium according to the invention comprising:
According to the invention, step (c), which may be an autoclaving stage, is advantageously carried out after mixing with maltose.
According to the invention, step (c) of heating the first mixture at the adjusted pH obtained in step (b) to a temperature of 110 to 130° C. for a period of 15 to 30 minutes, followed by cooling to a temperature which may be from 0° to 60° C., can be carried out by autoclaving, according to any technique and with any apparatus known to a person skilled in the art to sterilize a culture medium without chemically modifying its components.
According to the invention, the maltose monohydrate may be added in step (a), that is before step (c), or in step (d) or (f), that is after step (c), preferably in step (a).
According to the invention, step (e) can be carried out as step (c), that is to say by heating the second mixture obtained in step (d) to a temperature of 110° to 130° C. for a period of 15 to 30 minutes, followed by cooling at a temperature of 0° to 60° C., or by filtration at 0.2 μm.
According to the invention, the step (f) of mixing the first and second mixtures is preferably carried out in sterile medium, for example under a microbiological safety station. According to the invention, the method may also comprise a degassing step (g),
for example under N2H2CO2, for example lasting 15 minutes to 6 hours, following the volume to be degassed, for example for 2 to 6 hours for an industrial bioreactor. According to the invention, the degassing may also be passive degassing, for example lasting one night to several days, preferably in an anaerobic enclosure.
The present invention also relates to a method for culturing a bacterium comprising a seeding of a culture medium according to the invention by means of the bacterium, and a culture of the bacterium in the culture medium at a temperature allowing its growth.
A person skilled in the art knows the optimal bacterial growth temperatures or knows how to determine them, in particular from simple growth curves as a function of temperature. When they are intestinal bacteria, the culture temperature is generally 37° C.
According to the invention, the bacterium may be one of the aforementioned bacteria, for example an intestinal anaerobic bacterium, F. prausnitzii or an oxygen-sensitive bacterium or a strict anaerobic bacterium.
According to the invention, as the culture is for anaerobic microorganisms or even strict anaerobes, the culture is naturally carried out under anaerobic or strict anaerobic conditions. Anaerobic culture methods are known to a person skilled in the art and are applicable herein. Strict anaerobic bacteria can only be cultured in the absence of oxygen. This therefore requires limiting contact with ambient air. The culture medium can be incubated once inoculated in an oxygen-free environment, for example in an anaerobic chamber, which is a chamber of a Freiter chamber type, using gas mixtures to create the anaerobic conditions, for example N2 and/or CO2, or in an anaerobic jar or plastic pouch that use chemical catalysts to create the anaerobic conditions, that is an atmosphere of less than 1% O2 or even 0% O2.
Other advantages will become more apparent in light of the following examples, given by way of non-limiting illustration, with reference to the appended figures.
Table 1 below lists the components used in these example embodiments of the invention.
Table 2 below lists the various compositions of the components listed in Table 1 used in these example embodiments of the invention.
The method for producing the culture medium was as follows:
For example, in Hungate tubes: dissolving components 1 to 4 in a volume of distilled water which was chosen as a function of the desired final volume. Part of the volume of distilled water was set aside, in this example 1%. The volume of distilled water was 1 L, and the volume set aside was 100 mL. Components 1 to 4 were added, each in the amount making it possible to obtain the abovementioned concentrations when the culture medium is finalized. Several media were manufactured, with indicated variations of concentrations. A first mixture was therefore each composed of the components 1 to 4. The pH of this first mixture was then adjusted to 7.4 with 10 M of soda.
The first mixture was heated to a temperature of 120° C. for 20 minutes in an autoclave in order to sterilize it. It was then left to cool. During this time, the components 5 to 8 are dissolved in the rest of the distilled water, here 100 mL (each in sufficient quantity to obtain the abovementioned concentrations when the culture medium is finalized), in order to obtain the second mixture. This second mixture was then sterilized by filtration at 0.2 μm under a microbiological safety station.
The second mixture is then added to the first mixture and the assembly mixed to homogeneity under the microbiological safety station, then subjected to degassing by means of a mixture of H2CO2 in N2 (BlO-301, Air Liquide) for 4 hours.
For example in a bioreactor: compounds 1 to 4 are dissolved in distilled water and then this “basal” medium is placed in the bioreactor. The bioreactor is then autoclaved directly with the medium and the probes. Then, the filtered solution of vitamins and cysteine (compounds 5 to 8) is added to the bioreactor via a cannula under sterile conditions thanks to the use of a flame. Then, the medium is degassed directly in the tank for 30 minutes.
Various media were thus manufactured, including media 1 to 5 defined above.
Different media manufactured as in example 1 were seeded with 2% to 4% by preculture volume of Faecalibacterium prausnitzii. These volumes correspond to seeds making it possible to obtain initial concentrations of bacteria in cultures between 1E+06 and 1E+08 CFU/mL.
In these examples, the F. prausnitzii used is the DSM 17677 strain (Hauduroy et al. 1937, Duncan et al. 2002 emend. Fitzgerald et al. 2018), from the German Collection of Microorganisms and Cell Cultures GmbH (Germany).
Before being used, all the media and all the liquid solutions coming into contact with the bacterium were actively degassed by bubbling the gaseous mixture N2H2CO2 indoors for 4 h. This gas mixture was also used whenever it was necessary to establish or maintain anaerobiosis in a hermetic container or an enclosed space.
Once the media were seeded, they were placed in strict anaerobic culture at 37° C. in a Memmert oven, under the following conditions: anaerobiosis preserved by the Hungate tubes, 37° C. established owing to the oven, in a temperature bioreactor regulated by a bio-controller, anaerobiosis preserved by the passage of N2H2CO2.
Appended
The curves shown in
In this figure, it can be seen that the growth of F. prausnitzii is sustained and enters the exponential phase more rapidly when the cultures are seeded at 4%.
The curves shown in
In this Figure, it can be seen that the pH decreases rapidly during the exponential culture phase and stabilizes around a value of 5.3 in stationary phase, for both series of culture. However, the drop in pH begins more quickly for the series of culture carried out with a seeding rate of 4%.
The curves shown in
In this figure, it can be seen that the various media in accordance with the present invention that were tested made it possible to obtain about 2×109 CFU/mL during cultures in Hungate tubes and in a useful 0.5 L bioreactor. However, the series of culture carried out with a seeding rate of 4% has a shorter latency phase and enters an exponential phase more quickly than the series of culture carried out with a seeding rate of 2%. Still, the maximum CFU/mL values reached remain identical.
The curves shown in
In this
The curve shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2013684 | Dec 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2021/052397 | 12/17/2021 | WO |
