RU2778569C2 - Adaptation method - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to a method for adaptation of anaerobic bacterial strains in an oxidizing environment and its use in the development of new probiotics. A method for adaptation of anaerobic bacterial strains and selection of more oxygen-tolerant anaerobic bacterial strains is proposed, including stages of cultivation of the specified bacterial strains, using phase double induction of oxidative stress, using applied pressure and oxygen diffusion, and phase changing in the ratio of concentrations of antioxidant/oxidized variant for correction of a redox status. A method for the production of oxygen-adapted anaerobic bacterial strains, as well as oxygen-adapted bacterial strains used as probiotic are also proposed.

EFFECT: bacterial strains, which show an increased level of tolerance to the surrounding air compared to an initial or parent strain, which is not adapted to oxygen.

21 cl, 10 dwg, 4 tbl, 6 ex

Description

FIELD OF THE INVENTION

The present invention mainly relates to the cultivation of bacteria. More specifically, the invention relates to a method for adapting anaerobic microorganisms to an oxidizing environment and its use in the development of new probiotics. The present invention also relates to new (adapted) strains obtained by the methods of the invention.

BACKGROUND OF THE INVENTION

The adult gut microbiota consists of up to 100 trillion microorganisms, which is equivalent to ten total somatic and germ cells of the body. The genome population of gut microbes (the microbiome) can contain more than 150 times as many genes as a person's own genome and endows humans with physiological abilities that humans have not had to develop on their own. However, these communities remain largely unexplored, leaving almost completely unknown their impact on human development, physiology, immunity, nutrition, and health. The microbial community has an unlimited ability to influence host biology and is fundamental to the development of the human immune system, the ability to digest otherwise indigestible dietary polysaccharides, in addition to being a source for the formation of vitamins and hormones, etc. Microbial communities in the colon play a vital role in the regulation of gut health and the pathogenesis of several diseases.

Traditional microbiology focuses on the study of individual species as isolated units. However, many, if not most, have never been successfully isolated as viable samples for analysis, probably because their growth depends on a specific microenvironment not replicated under experimental conditions. Advances in DNA sequencing technologies have created a new field of research called metagenomics, enabling the comprehensive study of microbial communities, even those composed of uncultivated organisms. Instead of studying the genome of a single bacterial strain grown in a laboratory, the metagenomic approach allows the analysis of genetic material obtained from complete microbial communities collected from natural environments.

The human gut microbiome serves as a resource for beneficial microorganisms, potential probiotics. Despite the preventive and stimulatory role of the gut microbiota in diseases such as inflammatory bowel disease, cardiovascular disease, diabetes, and autism spectrum disorders, none of the available drugs contain new probiotics or so-called next-generation probiotics, as described in several metagenomic studies. research. Even if successfully isolated, many gut-isolated microbes require strict anaerobic growth conditions and cannot survive for more than a couple of minutes when exposed to ambient air. This complicates the development of stable and sustainable probiotic formulations. Thus, there is a need for more oxygen tolerant anaerobic microorganisms, and the present invention relates to a method for adapting anaerobic microorganisms, as well as new strains of microorganisms obtained by such methods.

SUMMARY OF THE INVENTION

The present invention relates to a method for adapting anaerobic microorganisms to an oxidizing environment, thus allowing such organisms to be relatively more oxygen tolerant when exposed to ambient air. It has the additional benefit of improved cultivation and storage of microorganisms, such as longer storage.

The present invention relates to a Human Intestinal Redox Simulation Model (SHIRM) in which stepwise dual induction of oxidative stress occurs with applied voltage and oxygen diffusion and stepwise alteration of the antioxidant/oxidized variant concentration ratio to correct redox status.

Oxygen is lethal to anaerobic microorganisms, so the dissolved oxygen used for oxidative stress (oxygen concentration) will increase but remain at sub-lethal levels when an antioxidant/oxidized variant pair is used to correct the redox status of the environment. Oxidative stress is also induced by applied voltage. Oxidative stress increases the tolerance of bacteria to an oxidizing environment and allows the viability of microorganisms in the ambient air for relatively longer periods of time.

Suitable sub-lethal oxygen concentrations (concentrations that do not kill all bacteria present, or, in other words, concentrations that kill some, but not all, bacteria) can easily be determined, for example, in such a way that at least some ( but generally not all) bacteria can survive and then be selected for the next stage of the methods. The surviving bacteria are then grown again, e.g., with increasing oxidative stress (e.g., by increasing the concentration of dissolved oxygen), so that again at least some (but usually not all) bacteria can survive and then can be choose for the next stage of the methods. This step of increasing oxidative stress can then be repeated again as many times as needed or desired to produce more oxygen tolerant microorganisms.

The present invention relates to a method for adaptation of anaerobic microorganisms and selection of more oxygen tolerant anaerobic microorganisms, comprising the steps of cultivating said microorganisms with stepwise dual induction of oxidative stress by applied voltage and oxygen diffusion and stepwise change in redox status, where, preferably, stepwise change redox status using a stepwise change in the ratio of antioxidant/oxidized variant concentrations to correct the redox status.

The present invention relates to a method for adaptation (or training) of anaerobic microorganisms and selection of more oxygen tolerant (for example, relatively more oxygen tolerant) anaerobic microorganisms, where microorganisms are cultivated with a combination of staged double induction of oxidative stress using applied voltage and oxygen diffusion and staged change the ratio of antioxidant/oxidized variant concentrations to correct the redox status.

The present invention further relates to a method for improved production of anaerobic microorganisms, wherein the microorganisms are cultured by combining the oxidation potential/applied voltage constant, oxygen diffusion, and the antioxidant/oxidized variant concentration ratio, thereby enabling selective pressure and obtaining a high yield of specific strains of microorganisms. Said culture is under optimal conditions, or in other words, the combination of oxidative potential/applied voltage constant, oxygen diffusion, and antioxidant/oxidized variant concentration ratio is optimized to produce anaerobic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The figure 1 shows the combination of oxidation-reduction of the lumen of the human intestine. Despite the presence of a continuous influx of oxygen through food intake and through diffusion from the intestinal mucosa, the cumulative redox potential of the intestine remains negative, approximately -300 mV.

Figure 2 shows the potential damage to oxygen sensitive enzymes caused by oxidative stress and the recovery mechanism.

Figure 3 shows the main enzymatic pathway used by the microbiota in the human gut.

Figure 4a

Custom-made 3-chamber Human Gut Redox Simulator (SHIRM).

Figure 4b

Scheme of electron flow from bacterial cells to electron acceptors in SHIRM. Redox potentials (reduction potential and oxidation potential) measured in (E _o ') volts.

Figure 4c

SHIRM scheme of intermediates involved in the transfer of electrons from a bacterial cell to electron acceptors, including redox potentials.

Figure 5

The main course of SHIRM operations.

Figure 6

Image of a SHIRM anode chamber connected to a small bed volume, approximately 100 ml, of an oxygen feeder.

Figure 7

SHIRM with air consumption: direct diffusion of oxygen from the air into the anode chamber via a side branch.

Figure 8

The course of SHIRM operations, 10 stages.

Figure 9

Selection of adapted strains.

Figure 10

Strategy for studying tolerance profiles of adapted strains to oxygen.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

DEFINITIONS

"Adaptation" is a dynamic evolutionary process, such as the result of natural selection, whereby an organism becomes more adapted to life in its natural environment. The process of adaptation (or training) of microorganisms can also preferably be carried out in a non-native/artificial/ in vitro environment, for example, by the methods of the present invention.

An "antioxidant" is an agent that prevents the formation of reactive oxygen, nitrogen, hydroxyl, and lipid species by scavenging free radicals or repairing or removing damaged molecules. Antioxidants can often act directly as reducing agents.

A "reductant" is a substance, element, or compound that loses (or "donates") an electron to another agent in a redox chemical reaction. Because the reducing agent loses electrons, it is said to be oxidized.

"Oxidized variants" are chemicals that cause the removal of an electron from other molecules. The oxidized variant is the oxidized form of the antioxidant.

"Redox mediators" or electron carriers are compounds that can undergo a reversible redox reaction and facilitate the transfer of electrons to and from bacteria.

The terms "applied voltage", "external voltage", "oxidation potential", "applied electrical oxidation potential", "applied electrode potential", "voltage" and "voltage potential" may be used interchangeably and refer to the voltage applied to the chambers. bioreactor.

"Oxygen diffusion" is the movement of oxygen molecules from high concentration to low concentration.

"Oxygen influx" is the amount of oxygen entering a chamber per unit of time, such as an anode chamber containing microorganisms.

The gut microbiota, together with dietary redox active compounds and electron donors such as carbohydrates and proteins, play a role in maintaining the reducing environment in the gut. This means that the intestinal microbiota is adapted to cope with the influx of dissolved oxygen in the intestinal lumen (figure 1). Dissolved oxygen at high concentrations can be lethal to a large number of gut microorganisms and the reducing environment helps them recover from oxidative damage (Figure 2). In more detail, Figure 3 shows the main enzymatic pathway used by the microbiota in the human gut. The disproportion of electrons during anaerobic growth is balanced by fermentation with the formation of a mixture of organic acids and the formation of gas. Some microorganisms donate electrons to extracellular electron acceptors such as nitrate, sulfate, or an insoluble metal complex. In addition, oxygen can act as an indirect electron acceptor, as some of the intestinal micro-organisms can consume oxygen in small amounts. Redox active compounds such as gallates, rutin, resveratrol, caffeine and flavins are present in common foods, probably in a form oxidized in the digestive tract, they can also act as electron acceptors (figure 1). The redox status of the intestine may affect the bioavailability of specific drugs or metabolites. Despite the presence of a continuous influx of oxygen through food intake and through diffusion from the intestinal mucosa, the cumulative redox potential of the intestine remains negative, approximately -300 mV. This oxygen deficient reducing environment promotes the growth of certain anaerobic microorganisms in the intestinal lumen.

High sensitivity to oxygen is a characteristic feature of many intestinal microorganisms, which makes it extremely difficult to cultivate and store these microorganisms for a long period of time. Another hurdle in isolating and culturing members of the gut microbiota for the development of new potential probiotics is the complexity of the gut environment in terms of different niches and habitats. The composition of the probiotic should be stable in the ambient air for several months, but this is difficult given the sensitivity of many intestinal microorganisms to oxygen. Some gut micro-organisms cross-feed from other micro-organisms and may also require some carrier growth factors, making these conditions difficult to mimic in vitro .

Faecalibacterium prausnitzii is an example of a potential probiotic with anti-inflammatory properties in various metabolic diseases. They make up over 5% of the bacteria in the human gut, making them one of the most abundant bacteria in the gut microbiota. F. prausnitzii produces relatively large amounts of butyrate, which plays an important role in intestinal physiology and is the main metabolite in the lumen of the large intestine. Butyrate also stimulates cell proliferation and promotes mucus secretion from the colonic mucosa. F. prausnitzii is an extremely oxygen sensitive bacterium and lives for only a few minutes when exposed to ambient air.

The present inventors have unexpectedly discovered a new way of adapting anaerobic microorganisms to oxygen, and in the present description describe a method of said adaptation to oxygen, thus allowing such organisms to become relatively more oxygen tolerant and thus stable and stable in storage and use as probiotics. strains and in probiotic formulations or compositions for exposure to mammals. The new method, in particular, will enable the development and use of F. prausnitzii as a probiotic strain with robust characteristics such that they are tolerant when produced on an industrial scale and able to adapt to the human intestinal environment under conditions of intestinal disorders, for which the main feature is oxidative stress.

The present invention relates to a method for adaptation of anaerobic microorganisms and selection of relatively more oxygen tolerant anaerobic microorganisms, where the microorganisms are cultured using a combination of stepwise dual induction of oxidative stress by applied voltage and oxygen diffusion and stepwise change in the ratio of antioxidant/oxidized variant concentrations to correct the redox status . Thus, the methods of the invention are generally carried out in vitro.

The dual induction of oxidative stress increases the tolerance of microorganisms to an oxidizing environment, thus allowing the microorganisms to be more, or relatively more, oxygen tolerant (eg, when exposed to ambient air). The term "microorganisms more (or relatively more) tolerant of oxygen (eg, when exposed to ambient air)" may refer to comparison with the initial strains (or parental strains) used in the adaptation methods of the invention.

Increased oxygen tolerance allows the viability of microorganisms in the ambient air for relatively longer periods of time, which is clearly an advantage.

An applied voltage or an applied external voltage is generally applied to an anode, such as a graphite, graphite felt, or carbon felt, or carbon fiber anode, for example, through a potentiostat, and increased step by step. To apply voltage to microorganisms, they are placed in a suitable anode chamber or other vessel, such as a bioreactor, to which voltage is applied. The circuit is closed, for example, by connecting the cathode chamber to the anode chamber, for example by means of one side branch of the anode chamber, preferably separated by an ion exchange membrane (eg, a cation selective membrane or a proton exchange membrane). The applied voltage can be increased (preferably increased in stages) up to a maximum of 0.6 V, more specifically 0.6 V relative to Ag/AgCl.

Typically, the methods involve the use of oxygen diffusion, for example, by supplying oxygen to the microorganisms. Oxygen supply (eg, oxygen supply through the anode chamber) can be maintained by purging the oxygen feeder with pure oxygen gas or air so as to achieve desired dissolved oxygen concentrations (in particular, desired concentrations of dissolved oxygen in contact with microorganisms). Dissolved oxygen will diffuse through the oxygen feed to the anode chamber, thus creating oxygen diffusion (or oxygen influx) in the manner described. The influx (or diffusion) of oxygen, for example, can be controlled by any suitable means, for example, by connecting an oxygen feed or other source of oxygen to the anode chamber, for example, another side branch of the anode chamber, separated, for example, by a variable partition (for example, a variable membrane, such as semi-permeable membrane) having different oxygen diffusion constants. Additionally or alternatively, the oxygen supply can be controlled by varying the volume of the oxygen feeder bed. For example, the figures show 100 ml and 250 ml oxygen feeders (see figures 6 and 4a). The oxygen feed can also be removed, allowing direct diffusion of oxygen into the anode chamber, as shown in Figure 7, for example. possible better controlled diffusion of oxygen from the oxygen feeder into the anode chamber.

In addition, reducing agents or antioxidants present in the environment will remove dissolved oxygen and thus affect oxygen diffusion kinetics and thus oxygen influx. For example, in the methods of the present invention, antioxidants (in particular, the amount or level of antioxidants) present in the microorganism culture medium can be used to remove oxygen and thus as a means to control the levels or amount or concentration of oxygen (dissolved oxygen) present in the culture of microorganisms. For example, a low (or lower) level of an antioxidant in a microorganism culture will typically mean that more oxygen is available (or more oxidative stress is occurring), while a high (or higher) level of an antioxidant will typically mean less oxygen available (or low oxygen levels) (or lower oxidative stress).

Any of these methods of controlling diffusion or oxygen influx, or a combination thereof, can easily be used to achieve the desired amount or concentration of dissolved oxygen in the microbial culture medium and thus achieve a step change (preferably a step increase) in oxygen diffusion, oxygen influx, or dissolved oxygen concentration (or amount or level of dissolved oxygen) used to induce oxidative stress in the methods of the present invention.

Thus, in the methods of the invention, the initial conditions are preferably low (or no) oxygen influx (or oxygen diffusion or oxygen concentration), and the methods include stepwise increase in oxygen influx (or oxygen diffusion or oxygen concentration), for example, up to high oxygen influx (or oxygen diffusion or oxygen concentration). The term "zero oxygen influx" (or oxygen diffusion or oxygen concentration) refers to 0 moles of oxygen present in the microbial culture medium (eg, an anode chamber). An example of low oxygen influx (or oxygen diffusion or oxygen concentration) refers to up to about 10 μM of dissolved oxygen present in the microbial culture medium (e.g., in the anode chamber), such as diffusing into the microbial culture medium/anode chamber per hour. An example of high oxygen influx (or oxygen diffusion or oxygen concentration) refers to about 50-250 μM of dissolved oxygen present in the microbial culture medium (e.g., in the anode chamber), such as diffusing into the microbial culture medium/anode chamber per hour.

In fact, oxidative stress achieved by oxygen influx (or oxygen diffusion or oxygen concentration) also depends on the presence of antioxidants in the growth medium. For example, if strong antioxidants are present, a high supply of oxygen will result in less oxidative stress. If very weak antioxidants are present in the environment, a small supply of oxygen can lead to high oxidative stress. Again, controlling these conditions to achieve a stepwise induction of oxidative stress is within the skill of one skilled in the art.

To control the initial concentration of dissolved oxygen (eg, to reduce the initial concentration of oxygen to a low (or zero) level), the growth medium can be degassed with respect to oxygen, for example, by purging with oxygen-free nitrogen.

An antioxidant is included in the culture medium to protect the microorganisms from lethal oxygen levels and to mimic the redox environment of the intestine. For example, the antioxidant may be cysteine, glutathione, ascorbic acid, dithiothreitol, or gallic acid. An antioxidant, such as cysteine or any other suitable antioxidant, will be used to influence or correct the redox potential or redox status. Many bacteria require cysteine as a source of sulfur. If cysteine is reduced, as in some of the methods of this invention, this will result in a reduction in the total sulfur content of the growth medium. To compensate for this reduction, an oxidized variant, such as cystine (in the case of cysteine), is added. Other oxidized variants (for example, in the case of the antioxidants mentioned above), if necessary, may be oxidized glutathione, dehydroascorbate, oxidized dithiothreitol or oxidized gallic acid. In addition, suitable redox mediators may also be included in the culture media.

Stepping the redox status, eg stepping the antioxidant/oxidized variant concentration ratio, is performed to correct the redox status of the growth medium. The redox potential can be calculated using the Nernst equation (a). It can be used for any suitable antioxidant and its oxidized version.

(a)

(a)

where:

E _h= redox potential (V)

E _o = Standard redox pair (Reductor/Antioxidant)/Oxidized variant

R=Universal Gas Constant=8.31451 J K ^-1 mol ^-1

F=Faraday constant=96.485 C/mol

T=Absolute temperature (K)

n=Number of electrons involved.

Thus, a person skilled in the art can easily calculate the redox potentials of any ratio of antioxidant/oxidized variant concentrations. As an example, the values of the redox potential (E _h ) calculated for various concentrations of the redox pair of cysteine/cystine are presented in table 1.

E _o for a pair of cystine/cysteine=0.25 V at pH 7.4, and n=2.

When using cysteine/cystine as the antioxidant/oxidized variant pair, the preferred initial cysteine concentration can be calculated according to reaction equation (b).

4R-SH+O ₂ →2R-SS-R+H ₂ O (b)

According to equation (b), 4 moles of cysteine (R-SH) react with one mole of oxygen to form 2 moles of cystine (R-SS-R).

The concentration of dissolved oxygen in water at 25°C is approximately 200 μM, and if the growth medium is not purged with nitrogen before the experiment, approximately 800 μM cysteine will react with 200 μM oxygen to form 400 μM cystine.

Thus, in the case of a growth medium containing 8 mM cysteine, an initial redox potential of approximately -223 mV will be observed. However, complete oxygen degassing in the growth medium using oxygen-free nitrogen results in a redox potential of approximately -283 mV, close to the intestinal lumen redox (approximately -300 mV). Thus, if a cysteine/cystine pair is used, the preferred initial concentration of cysteine (or other antioxidant) is 8 mM. This is an example of a high antioxidant concentration when a cysteine/cystine pair is used, however, it should be understood that equivalent high antioxidant concentrations for other pairs can be easily calculated. In addition, the preferred initial concentration of cystine (or other oxidized variant) is 0 mm or otherwise low concentration to achieve the desired initial redox potential. It is also preferred to completely degas the growth medium used from oxygen (or completely remove oxygen from the growth medium). In other words, the preferred conditions are chosen to approximate as closely as possible the intestinal lumen redox potential of -300 mV as the initial conditions of the method. In the implementation of the methods, it is preferable to perform a stepwise decrease in the concentration of the antioxidant in combination with a stepwise increase in the concentration of the oxidized variant for an overall stepwise increase in redox potential. In particular, for cysteine/cystine (or another antioxidant/oxidized variant pair) it may be a stepwise decrease in the concentration of cysteine by 1 mM and an increase in the concentration of cystine by 1 mM. However, other stepwise increases/decreases can also be used, provided that the stepwise change in redox status is carried out.

This principle can be adapted to any suitable antioxidant/oxidized variant pair. When the antioxidant concentration decreases, it is balanced by an equivalent amount of the corresponding oxidized variant.

The method, in particular, can be carried out in a suitable bioreactor, and, as a rule, it includes several stages of subculture of the bacterial culture (for example, in new culture media). In some preferred embodiments of the invention, each step of stepping or inducing as described herein, such as stepping dual induction of oxidative stress with applied voltage and oxygen diffusion, and stepping the antioxidant/oxidized variant concentration ratio to correct redox status, for example, each step of stepping down the antioxidant concentration in combination with stepping up the concentration of oxidized variant, stepping up applied voltage, and stepping up oxygen influx (or oxygen diffusion or oxygen concentration) may include reseeding. The term "reseeding" in the present specification may also refer to a subculturing step or a culture step.

First, a single colony of bacteria can be seeded in suitable culture media, as is known in the art, and allowed to grow until the culture reaches stationary phase. The initial conditions may be one or more or all of low oxygen influx (or oxygen diffusion or oxygen concentration), preferably zero oxygen influx (or oxygen diffusion or oxygen concentration), low applied voltage, preferably 0.1 V, more specifically, 0.1 V relative to Ag/AgCl, high concentration of antioxidant (see Nernst equation above), low concentration or no concentration of oxidized variant (see Nernst equation above). In the case of subsequent passages, a stepwise decrease in the concentration of antioxidant, an increase in the oxidized variant, an increase in the applied voltage, and an increase in oxygen influx (or oxygen diffusion or oxygen concentration) can be carried out.

If microbial growth is significantly reduced, conditions can be kept constant at the optimized (or end) conditions reached, exemplified by 3 mM cysteine, 5 mM cysteine, 0.6 V vs. Ag/AgCl and 0.2 nmol⋅mL^-onemin^-one, respectively (see figures 5 and 8) to allow the strains to adapt. In other words, when a step change results in a significant reduction in microbial growth, the step change is terminated and the microorganisms are cultured for one or more steps using constant conditions (or optimized or end conditions) corresponding to the conditions reached by the end of the step change. These constant stages allow strains to adapt. After repeated seedings under constant conditions when the strain has adapted, in some embodiments, seedings can be continued using one or more (or preferably all) of an additional incremental reduction in antioxidant concentration, increase in oxidized variant, increase in applied voltage, and increase in oxygen supply (or oxygen diffusion or oxygen concentration) (see figure 5).

All subcultures obtained at each stage of the method can be analyzed simultaneously to select the most adapted strain, or the subculture can be analyzed immediately after production to decide whether it is necessary to continue crops or not. If the subculture is not adapted, then crops should be continued, otherwise, if the strain is adapted, you can choose this strain and stop the implementation of the method (see, for example, figure 8). For example, the obtained subcultures can be analyzed for oxygen tolerance and if they exhibit the desired level of oxygen tolerance, for example, an increased (preferably significantly increased) level of oxygen tolerance compared to the corresponding control strain (for example, initial or parent strains) or sufficient level of stability (eg storage time) when exposed to oxygen (eg in ambient air), then the strain is adapted and can be selected. Oxygen tolerance can be measured by assessing the growth of microorganisms under aerobic conditions (eg, exposure to ambient air). For example, it is possible to measure the number of colony forming units (CFU) if the strains are exposed to oxygen, for example in the form of a CFU/ml measurement. Example adapted (oxygen tolerant) strains will have at least 1×10 ² or 1×10 ³ cfu/ml, preferably at least 1×10 ⁴ or 1×10 ⁵ cfu/ml, when exposed to oxygen, e.g. ambient air. The corresponding method is described in the examples (see, for example, table 3). If the strain is not adapted, then crops can be continued.

Thus, in preferred embodiments of the invention, the initial conditions are selected from one or more, or all, of: 8 mM cysteine (or an equivalent concentration of an alternative antioxidant), 0 mM cystine (or an alternative oxidized variant), an applied voltage of 0.1 V, and zero or low oxygen supply. In such embodiments, incremental changes can be continued until one or more, or all, of: a minimum cysteine concentration of 3 mM (or an equivalent alternative antioxidant concentration), a maximum cystine concentration of 5 mM (or an equivalent concentration of an alternative oxidized variant), a maximum applied voltage of 0 .6 V and high oxygen supply.

Thus, in the preferred methods of the invention, the applied voltage does not exceed 0.6 V and/or the cysteine concentration is not less than 3 mM and/or the cystine concentration does not exceed 5 mM and/or the concentration of dissolved oxygen in the culture medium is maintained at sublethal concentration.

As can be seen from the above description, in some embodiments of the invention, after completion of the stepwise changes, the method includes one or more additional stages of cultivation (subculturing) of these microorganisms in the final (optimized or constant) conditions of applied voltage, oxygen diffusion (oxygen influx or oxygen concentration) and ratio of antioxidant/oxidized variant concentrations achieved after incremental changes (see, for example, figure 8).

In other embodiments, the implementation of the method further includes subsequent stages, which carry out further phased changes (see, for example, figure 5).

The number of incremental steps to be implemented can be determined by one skilled in the art by monitoring the behavior of the microorganisms or screening the microorganisms (eg, as described elsewhere herein) while they are being adapted. For example, preferred embodiments of the methods of the present invention may include up to 5 or 6 steps, such as 3, 4, 5, or 6 steps, or at least 5 steps, such as up to 10 steps, such as 7 , 8, 9 or 10 stages.

The total number of steps, e.g. cultivation steps (including step change steps and steps to be carried out under constant or optimized conditions), to achieve adaptation of anaerobic microorganisms and selection of more oxygen tolerant anaerobic microorganisms can be determined by one skilled in the art by monitoring the behavior of microorganisms or screening microorganisms (for example, as presented elsewhere in the present description) while they are being adapted. For example, preferred embodiments of the methods of the present invention may include up to 6, 7, 8, 9, 10, or 12 steps, or up to 15, 20, 25, 30, or 35 steps.

In methods involving culture steps to be carried out under constant or optimized conditions, the number of steps, e.g., culture steps to be carried out, can be determined by one skilled in the art by monitoring the behavior of microorganisms or screening microorganisms (e.g., as described elsewhere herein). ) while they are undergoing adaptation. For example, preferred embodiments of the methods of the present invention may include up to 3, 4, 5, 6, 7, 8, 9, 10, or 12 steps, or up to 15, 20, 25, 30, or 35 steps, performed under constant or optimized conditions.

In some embodiments, adaptation of the anaerobic microorganisms and selection of more oxygen tolerant anaerobic microorganisms can be achieved by the end of these stages performed under constant or optimized conditions (or in fact, sometimes by the end of the step change stages). Thus, a person skilled in the art can easily determine the number of such steps to be carried out, for example, by monitoring the achievement of a significant, or sufficient, or maximum adaptation (tolerance to oxygen).

The method can also be used to obtain an adapted and selected strain (for example, to obtain or grow a microorganism selected by this method). In such methods, culturing conditions are kept constant under optimal conditions, for example, optimized (or constant) conditions defined by the methods of the invention described above, for example, 3 mM antioxidant, 5 mM oxidized variant, 0.6 V and 0.2 nmol⋅ml ^{- 1} min ^-1 . This results in a high yield of the selected bacterial strain.

The method is particularly carried out in a bioreactor called "Human Gut Redox Simulation Model" (SHIRM), Figures 4a, b and c. In SHIRM, dual oxidative stress is achieved by external voltage (applied voltage) and oxygen diffusion (oxygen influx) and the redox potential/redox status of the system is controlled by the antioxidant/oxidized variant pair. Because oxygen is lethal to cultured organisms, dissolved oxygen will increase but remain at a sub-lethal concentration, while a stepwise change in the ratio of antioxidant/oxidized variant concentrations is used to correct the redox status of the environment (see, for example, figure 5 or figure 8, which provide examples of cysteine/cystine concentrations that can easily be adapted to other antioxidant/oxidized variant pairings). Oxidative stress, through applied voltage and oxygen diffusion, increases the bacteria's tolerance to the oxidative environment, and the cells are re-exposed to new oxidative conditions (eg, figure 5 or figure 8). Adapted or improved strains are screened for oxygen tolerance. Strains can also be screened for metabolic characteristics such as fatty acid profile (e.g. butyrate production in the case of butyrate-producing bacteria such asFaecalibacterium prausnitzii), and evaluation of growth kinetics for selection of the most adapted strain. Thus, adapted or improved strains are adapted for better tolerance (e.g. than that of the respective parent or parent organisms) to an oxygen-containing environment or for better tolerance to stressful conditions such as exposure to ambient air (oxidative stress) or, for example, bile salts. acids. Thus, if, for example, the strains areFaecalibacterium prausnitzii,choose strains in which metabolic characteristics, such as production of butyrate or other fatty acids, are retained or improved, but the strains exhibit improved oxygen tolerance (e.g., compared to the corresponding initial or parental strainFaecalibacterium prausnitzii).

In a preferred embodiment, the present invention relates to a method for adaptation of anaerobic microorganisms and selection of relatively more oxygen tolerant anaerobic microorganisms, where the microorganisms are cultured using a combination of stepwise dual induction of oxidative stress by applied voltage and oxygen diffusion and stepwise change in the concentration ratio of antioxidant/oxidized variant to correct redox status.

In another preferred embodiment, the method for adapting anaerobic microorganisms to oxygen is carried out using a bioreactor called the Human Gut Redox Simulation Model (SHIRM) and includes, but is not limited to, the following steps:

a) Inoculation of a single colony of bacteria in culture media (as is known in the art) including suitable redox mediators and reducing agents to protect the bacterial cells from lethal oxygen concentrations and to mimic the redox environment of the gut.

b) When the culture reaches the late exponential phase, one part of the culture can be used for analysis and the other part can be used for possible passage to the SHIRM reactor.

c) Inoculation of the culture in a SHIRM reactor containing the same culture media used in the primary isolation and routine culture of the bacterial strain. The growth medium is now called "Shirm Bed Culture Medium" (SBCM).

I. The concentration of the reducing agent/antioxidant, as non-limiting examples, cysteine, glutathione, ascorbic acid, dithiothreitol and gallic acid, reduced and balanced by an equivalent amount of the corresponding "oxidized variant", as non-limiting examples, cystine, oxidized glutathione, dehydroascorbate, oxidized dithiothreitol and oxidized gallic acid.

II. The applied electrical oxidation potentials are maintained by means of an external voltage, for example on an anode made of graphite, graphite felt, or carbon felt, or carbon fiber by means of a potentiostat. The circuit is closed, for example, by connecting the cathode chamber to one side branch of the anode chamber, separated by a proton exchange membrane.

III. The influx of oxygen is controlled by connecting an oxygen feeder to another side branch of an anode chamber separated by a variable baffle having different oxygen diffusion constants. The current influx of oxygen is measured, for example, using a dissolved oxygen sensor Clark, see figure 4A.

IV. The supply of oxygen is maintained by flushing the oxygen feeder with pure oxygen gas or air, and the desired dissolved oxygen concentrations are achieved following Henry's gas solubility law. The dissolved oxygen then diffuses through the oxygen feeder into the anode chamber.

V. In addition, the oxygen supply is controlled by changing the volume of the oxygen feeder bed. The oxygen feeder can also be removed to allow direct diffusion of oxygen into the anode chamber.

VI. The membrane wall between the anode chamber and the oxygen feeder can be further coated with mucin agar to allow better controlled diffusion of oxygen from the oxygen feeder into the anode chamber.

VII. SHIRM contains Shirm Bed Culture Media (SBCM) purged with oxygen-free nitrogen to remove dissolved oxygen.

VIII. The SHIRM bioreactor is turned on until the culture reaches the stationary phase.

d) One part of the first subculture is used for inoculation in the SHIRM reactor.

e) Start transferring to the SHIRM reactor having stepwise reduced antioxidant concentration, increased oxidized variant concentration, and increased applied voltage (maximum 0.6 V). Less antioxidant will remove less oxygen, so more oxygen will be available and the cells will experience more oxidative stress. The oxygen feeder additionally regulates the diffusion or influx of oxygen.

This step is repeated until growth decreases.

f) Keeping all conditions constant, repeat passages and analyze the new subculture for comparative metabolic characteristics, growth kinetics, stability and tolerance. If the results of the analysis are satisfactory, then this subculture is selected, and if not, then step f) is repeated.

In such methods, each subculture can be assayed prior to subsequent subculture (or assayed after each step) and the process terminated if the strain is suitably adapted to oxygen. Then choose this strain. In such methods, up to about 30 subcultures, or up to about 10 subcultures (or other number of subcultures as described elsewhere herein), may be analyzed before terminating the method.

If necessary, one or more of the above stages can be used in any of the methods according to the invention, as presented in the present description.

In another preferred embodiment, approximately 30 subcultures are obtained (see, for example, figure 5), or approximately 10 subcultures are obtained (see, for example, figure 8), or any number of subcultures are obtained (as presented elsewhere in the present description), and then all of them are analyzed to find the most suitable strain. Thus, it is not necessary to analyze subcultures after each stage, instead all analyzes can be carried out simultaneously, for example, to assess which subculture is most adapted or tolerant to oxygen. The method for adaptation of anaerobic microorganisms to oxygen is carried out using a bioreactor called "Human Gut Redox Simulation Model" (SHIRM), and it includes, as non-limiting examples, the following steps:

a) Inoculation of a single colony of bacteria in conventional culture media, as known in the art, including suitable redox mediators and reducing agents to protect bacterial cells from lethal oxygen concentrations and to mimic the redox environment of the intestine.

b) When the culture reaches the late exponential phase, one part of the culture is used for inoculation in the SHIRM reactor and the other part is left for analysis.

c) Inoculation of the culture in a SHIRM reactor containing the same culture media used in the primary isolation and routine cultivation of the bacterial strain. The growth medium is now called "Shirm Bed Culture Medium" (SBCM).

I. The concentration of the reducing agent/antioxidant, as non-limiting examples, cysteine, glutathione, ascorbic acid, dithiothreitol and gallic acid, reduced and balanced by an equivalent amount of the corresponding "oxidized variant", as non-limiting examples, cystine, oxidized glutathione, dehydroascorbate, oxidized dithiothreitol and oxidized gallic acid.

II. The applied electrical oxidation potentials are maintained by means of an external voltage, for example on an anode made of graphite, graphite felt, or carbon felt, or carbon fiber by means of a potentiostat. The circuit is closed, for example, by connecting the cathode chamber to one side branch of the anode chamber, separated by a proton exchange membrane.

III. The influx of oxygen is controlled by connecting an oxygen feeder to another side branch of an anode chamber separated by a variable baffle having different oxygen diffusion constants. The current influx of oxygen is measured, for example, using a dissolved oxygen sensor Clark, see figure 4A.

IV. The supply of oxygen is maintained by flushing the oxygen feeder with pure oxygen gas or air, and the desired dissolved oxygen concentrations are achieved following Henry's gas solubility law. The dissolved oxygen then diffuses through the oxygen feeder into the anode chamber.

V. In addition, the oxygen supply is controlled by changing the volume of the oxygen feeder bed. The oxygen feeder can also be removed to allow direct diffusion of oxygen into the anode chamber.

VI. The membrane wall between the anode chamber and the oxygen feeder can be further coated with mucin agar to allow better controlled diffusion of oxygen from the oxygen feeder into the anode chamber.

VII. SHIRM contains Shirm Bed Culture Media (SBCM) purged with oxygen-free nitrogen to remove dissolved oxygen.

VIII. The SBCM used for the first seed can then be referred to as SBCM X,Y/V_Z, where X refers to the concentration of antioxidant (mM), Y refers to the concentration of the oxidized variant (mM), and V_Z refers to applied voltage relative to Ag/AgCl (V). The SHIRM bioreactor is turned on until the culture reaches the stationary phase.

IX. The culture obtained after the first incubation may be referred to as "population P1".

d) The next seeding in the SHIRM reactor is started with a reduced antioxidant concentration, an increased oxidized variant concentration, and an increased applied voltage. Less antioxidant will remove less oxygen, so more oxygen will be available and the cells will experience more oxidative stress. The oxygen feeder additionally regulates the diffusion or influx of oxygen.

e) Subcultures continue until about the 6th subculture is obtained, see for example Figures 5 and 8 (when the combined effect of antioxidants/redox mediators, oxygen supply and stress significantly affects (reduces) growth).

f) The applied voltage will generally not be raised above 0.6 V with respect to Ag/AgCl due to excess voltage. Higher potentials can cause electrode fouling and, in addition, lethal oxidation of microbial cytochromes, for example cytochrome a3 has a reduction potential of approximately +0.385 V relative to SHE.

g) Keeping all conditions constant, repeated crops were made until about the 27th subculture (see, for example, figure 5) or about the 10th subculture (see, for example, figure 8), a more accurate number of subcultures (to increase the likelihood adaptation) to adaptation of the strain to the conditions, while fresh medium was used for each subculture.

h) After the 27th subculture, seeding was continued until about the 30th subculture, as shown in figure 5. Alternatively, after the 10th subculture, the crops were stopped, as shown in figure 8.

i) Upon receipt of all subcultures, they are analyzed for comparative metagenomic, metabolic characteristics, growth kinetics, stability and tolerance to oxidative stress to select the most adapted strain.

If necessary, one or more of the above stages can be used in any of the methods according to the invention, as presented in the present description.

In yet another embodiment, a method for adapting anaerobic microorganisms to oxygen is carried out using a bioreactor called the Human Gut Redox Simulation Model (SHIRM) and includes, but is not limited to, the following steps:

a) Inoculation of a single colony of bacteria in conventional culture media, as is known in the art. Cysteine is used to mimic the redox potential of the colon in the growth medium, and resazurin is used as a redox indicator.

b) When the culture reaches the late exponential phase, one part of the culture is used for inoculation in the SHIRM reactor and the other part is left for analysis.

c) Inoculation of the culture in a SHIRM reactor containing the same culture media used in the primary isolation and routine cultivation of the bacterial strain. The growth medium is now called "Shirm Bed Culture Medium" (SBCM).

I. The concentration of the reducing agent/antioxidant, cysteine, is reduced and balanced by an equivalent amount of the corresponding "oxidized variant", cystine.

II. The applied electrical oxidation potentials are maintained by means of an external voltage, for example on an anode made of graphite, graphite felt, or carbon felt, or carbon fiber by means of a potentiostat. The circuit is closed, for example, by connecting the cathode chamber to one side branch of the anode chamber, separated by a proton exchange membrane.

III. The influx of oxygen is controlled by connecting an oxygen feeder to another side branch of an anode chamber separated by a variable baffle having different oxygen diffusion constants. The current influx of oxygen is measured, for example, using a dissolved oxygen sensor Clark, see figure 4A.

IV. The supply of oxygen is maintained by flushing the oxygen feeder with pure oxygen gas or air, and the desired dissolved oxygen concentrations are achieved following Henry's gas solubility law. The dissolved oxygen then diffuses through the oxygen feeder into the anode chamber.

V. In addition, the oxygen supply is controlled by changing the volume of the oxygen feeder bed. The oxygen feeder can also be removed to allow direct diffusion of oxygen into the anode chamber.

VI. The membrane wall between the anode chamber and the oxygen feeder can be further coated with mucin agar to allow better controlled diffusion of oxygen from the oxygen feeder into the anode chamber.

VII. SHIRM contains SBCMs purged with oxygen-free nitrogen to remove dissolved oxygen.

VIII. The SBCM used for the first seeding can then be referred to as SBCM 8.0/V _0.1 where the former denotes the cysteine concentration while the latter denotes the cystine concentration in mM and "V" denotes the applied voltage against Ag/ AgCl. The SHIRM bioreactor is turned on until the culture reaches the stationary phase at an oxidative potential of +100 mV.

IX. The culture obtained after the first incubation may be referred to as "population P1".

d) The next seeding in the SHIRM reactor is started with a reduced cysteine concentration, an increased cystine concentration, and an increased applied voltage, see figure 5 and figure 8. Less antioxidant will remove less oxygen, thus more oxygen will be available and the cells will experience more oxidative stress. The oxygen feeder additionally regulates the diffusion or influx of oxygen.

e) Subcultures continue until about the 6th subculture is obtained. At this stage, the antioxidant concentration is reduced to 3 mM and the "oxidized variant" concentration is increased to 5 mM, while the oxidation potential is 0.6 V (relative to Ag/AgCl). The potential voltage will not be raised above 0.6 V with respect to Ag/AgCl due to excess voltage. Higher potentials can cause electrode fouling and, in addition, lethal oxidation of microbial cytochromes, for example cytochrome a3 has a reduction potential of approximately +0.385 V relative to SHE.

f) Keeping all conditions constant, re-plating was carried out until about the 27th subculture (figure 5) or the 10th subculture (figure 8) was obtained, with fresh medium used for each subculture.

g) In the situation shown in figure 5, after the 27th subculture, the crops were continued until about the 30th subculture, as shown in figure 5. Alternatively, for example, as shown in figure 8, crops were stopped after the 10th subculture.

If necessary, one or more of the above stages can be used in any of the methods according to the invention, as presented in the present description.

A stepwise change in the ratio of antioxidant/oxidized variant concentrations to correct the redox status corresponds to the Nernst equation. The following is an example for a cysteine/cystine redox pair:

E _h =E _o +RT/2Fln([Cystine]/[Cysteine] ² ) (a)

Where:

E _h= redox potential (V)

Eo=Standard redox potential of a pair of cystine/cysteine=0.25 V at pH 7.4

R= Universal gas constant=8.31451 J K ^-1 mol ^-1

F= Faraday constant=96.485 C/mol

T=Absolute temperature (K).

The values of the redox potential (E _h ) calculated for various concentrations of the redox pair of cysteine/cystine are presented in table 1.

Table 1

Cysteine cystine E _h mm mV 7.2 0.4 -223 Oxygenated environment 7.995 0.005 -283 Oxygen-deprived growth medium obtained by nitrogen purge 7 one -211 6 2 -198 5 3 -188 four four -178 3 5 -168 2 6 -155 one 7 -135 0.01 eight -13

4R-SH+O ₂ →2R-SS-R+H ₂ O (b)

According to equation (b), 4 moles of cysteine (R-SH) react with one mole of oxygen to form 2 moles of cystine (R-SS-R).

The concentration of dissolved oxygen in water at 25°C is approximately 200 μM, and if the growth medium is not purged with nitrogen before the experiment, approximately 800 μM cysteine will react with 200 μM oxygen to form 400 μM cystine. Thus, in the case of a growth medium containing 8 mM cysteine, an initial redox potential of approximately -223 mV will be observed. However, complete degassing of oxygen in the growth medium using oxygen-free nitrogen results in a redox potential of approximately -283 mV, close to the redox potential of the intestinal lumen (approximately -300 mV).

The present invention further relates to examples of adapted microorganisms produced by the methods of the present invention.

Thus, a further aspect of the invention relates to a microorganism (adapted microorganism), for example, a strain of a microorganism obtained, obtained, identified or selected by the methods of the invention. Such strains may be a probiotic microorganism or a bacterial strain. In the context of the invention, the term "probiotic" refers to microorganisms conferring health benefits when consumed. For example, the Food and Agriculture Organization of the United Nations defines probiotics as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." Such microorganisms or strains may be isolated strains or pure cultures and will not match natural microorganisms or strains, as they are subjected to the adaptation methods of the invention. Preferred microorganisms or strains are F. prausnitzii.

Examples of adapted strains obtained, etc. adaptation methods of the present invention are strains of F. prausnitzii, referred to in the present description as TCS1, OCS-1 and OCS-2. These strains were deposited in accordance with the Budapest Treaty with the DSMZ (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on October 12, 2016 under registration numbers DSM 32380, DSM 32378 and DSM 32379 , respectively.

Microorganisms or strains obtained, etc. the methods of the invention (eg, one or more deposited strains) may be in the form of a compound (agent) or composition, eg, a pharmaceutical compound or composition or a nutritional compound or composition.

Thus, the present invention also relates to a composition or formulation comprising:

(i) a microorganism or strain obtained, produced, identified or selected by the method of the invention, or a microorganism or strain of the invention otherwise defined herein (eg, one or more deposit strains); and

(ii) at least one additional component selected from the group consisting of a carrier, diluent, or excipient (eg, a pharmaceutically acceptable carrier, diluent, or excipient), a food or nutritional supplement, or an additional therapeutic or nutritional agent. Thus, said compositions can be formulated as pharmaceutical compositions or as food compositions, for example as a food product.

The present invention also relates to the therapeutic use of microorganisms, strains, compositions and compositions according to the invention, as defined in the present description (for example, one or more deposited strains).

Suitable route of administration and composition of microorganisms, strains, compositions, formulations, etc. selected depending on the location of the disease. Preferred routes of administration are oral or rectal, however, intravenous or intramuscular injections may be equally suitable.

A person skilled in the art can readily select or determine appropriate dosages of the microorganisms, strains, compositions and formulations of the invention as defined herein, depending on the disorder being treated, the route of administration and the formulation in question. For example, the dosage and administration schedule is chosen such that the microorganisms, strains, compositions, or compositions of the invention administered to an individual can provide a therapeutic or health benefit. For example, you can use daily doses of microorganisms from 10 ⁴ to 10 ¹² , for example, from 10 ⁵ to 10 ¹⁰ , or from 10 ⁶ to 10 ⁸ , or from 10 ⁸ to 10 ¹⁰ cfu of bacteria.

Thus, the present invention relates to products or compositions or compositions or kits containing more oxygen-tolerant anaerobic microorganisms obtained from etc. methods of the present invention or otherwise defined in the present description. Advantageously, such products have an extended shelf life or a longer shelf life.

Preferred products or compositions contain frozen, lyophilized or dried bacteria (see also examples) and are preferably in a single dose format such as capsules, tablets or gels. Appropriate doses (eg, in the form of bacterial counts or CFUs) for use in such a product, etc. described elsewhere in the present description and in the examples. Such products may also include other ingredients, etc., such as preservatives (eg glycerol), stabilizers, gelling agents and/or cryoprotectants. In some embodiments, such additional components are non-natural agents.

If reduced concentrations, levels, or increases are mentioned herein, then preferably, such reduction (and, in fact, other reduction or negative effects, as indicated elsewhere in the present description) is a measurable decrease, more preferably a significant decrease, preferably - a statistically significant decrease, for example, with a probability of ≤0.05, when compared with the corresponding level or value.

When reference is made herein to increased concentrations, levels, tension, tolerance or growth, etc., such as a stepwise increase or increase in oxidative stress, oxygen influx (or diffusion or concentration), or oxygen tolerance, then preferably such the increase (and indeed other increase or benefits as defined elsewhere herein) is a measurable increase, more preferably a significant increase, preferably a statistically significant increase, e.g. appropriate level or value.

In fact, if significant changes are given in the present description, it is preferred that such changes are statistically significant changes, for example, with a probability of ≤0.05, when compared with the corresponding level or value.

The following are some examples of the invention, intended not to limit the invention in the present description, but to demonstrate in detail practical examples of how the invention can be used.

EXAMPLES

EXAMPLE 1

Adaptation of anaerobic microorganism to an oxidized environment

The Faecalibacterium prausnitzii strain was isolated from the feces of a healthy volunteer by microbiological pure culture under strict anaerobic conditions (5% H ₂ , 15% CO ₂ and 80% N ₂ ) using a chamber from Coy and was named FBT-22 (DSM 32186). (FBT-22 was deposited in accordance with the Budapest Treaty with DSMZ (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on October 20, 2015 under registration number DSM32186). The conventional culture medium used for isolation contains the following (g/L); yeast extract: 2.5; caseton: 10; glucose: 4.5; sodium chloride: 0.9; potassium hydrogen orthophosphate: 0.45; potassium dihydrogen orthophosphate: 0.45; ammonium sulfate: 1.32; sodium bicarbonate: 4 g; cysteine: 1, resazurin: 0.001; hemin: 0.01. The Vitamin Blend contains: 10mcg biotin, 10mcg cobalamin, 30mcg p-aminobenzoic acid, 50mcg folic acid, and 150mcg pyridoxamine. The final concentrations of short chain fatty acids (SCFA) in the medium were 33 mM acetate, 9 mM propionate and 1 mM each of isobutyrate, isovalerate and valerate. All components were added aseptically while the tubes were blasted with CO ₂ . Heat-labile vitamins were sterilized by filtration using a 0.22 μm filter and added after autoclaving the medium to obtain a final concentration of 0.05 μg.ml ^-1 thiamine and 0.05 μg.ml ^-1 riboflavin. The final pH of the medium was adjusted to 7.2±0.2 using 1N NaOH or 1N HCl. The media were autoclaved at 100 kPa, 121°C for 15 min. Cysteine was used to mimic the redox potential of the colon in the growth medium, and resazurin was used as a redox indicator. The inoculum for the SHIRM bioreactor was prepared by seeding a single colony in 7 ml of culture medium. After 12-16 hours of incubation at 37°C, the culture reached a late exponential phase (OD ₆₀₀ ~0.7). Two tubes with approximately 0.5 ml of the main culture were stored in 20% glycerol at -80°C and were designated as FFR. These FFR stocks were referred to as FFR-M1.1 and FFR-M1.2. FFRM1.1 and directly inoculated into 7 ml culture medium without thawing. Seeds were incubated anaerobically for 12-16 hours at 37° C. when the culture reached the late exponential phase (OD ₆₀₀ ~0.7), 2.5 ml of culture was seeded in a 250 ml SHIRM bioreactor. A proportion of this main culture was kept in duplicate (as above) and designated as FFR-M2.1 and FFR-M2.2. In the event of any contamination or failure, the experiment can be continued using appropriate FFRM cultures. The composition of the SHIRM Bed culture medium (SBCM) is the same as that used for the primary isolation of the FBT-22 strain (DSM 32186); subsequently, the cysteine concentration was reduced and balanced with an equivalent amount of cystine. The applied electrical oxidation potentials were maintained by means of an external voltage across the graphite anode (8.5 cm x 0.25 cm x 2.5 cm) using a potentiostat. The circuit was closed by connecting the cathode chamber to one side branch, separated by a proton exchange membrane. The oxygen supply was controlled by connecting an oxygen feeder to an anode chamber separated by a variable partition having different oxygen diffusion constants. Current oxygen influx was measured using a Clark dissolved oxygen sensor. The influx of oxygen is controlled by flushing the oxygen feeder with pure oxygen diffusing into the anode chamber through a semi-permeable membrane. The diffusion coefficients (D _Om ) and mass transfer constants (K _Om ) of oxygen for the membrane are 2.4×10 ^-6 cm ² /s and 1.3×10 ^-4 cm/s, respectively. In addition, the oxygen influx was controlled by varying the volume of the oxygen feeder bed, for example, 250 ml or 100 ml, as shown in figure 6. The oxygen feeder can also be removed to allow direct diffusion of oxygen into the anode chamber, as shown in figure 7. Coating membrane septa with mucin agar additionally allows you to control the diffusion of oxygen into the anode chamber. Mucin agar was obtained by dissolving and autoclaving the following components (g/l): agar: 2; NaCl: 8; KCl: 0.2; Na ₂ HPO ₄ : 1.42; _KH2PO4 : _0.24 ; type II mucin: 5. Media autoclaved at 100 kPa, 121°C for 15 min. SHIRM contains SBCM purged with oxygen-free nitrogen for 15 minutes to remove dissolved oxygen. The final concentration of cells in the SHIRM reactor was adjusted to approximately OD ₆₀₀ ~0.005.

The SBCM used for the first seeding of FFR-M2.1 was then designated as SBCM 8.0/V _0.1 where the first of the numbers ("8") is the cysteine concentration (mM), the last ("0") is the cystine concentration (mM), and "V" means the applied voltage relative to Ag/AgCl. Each experiment will start with zero oxygen influx and oxygen diffuses out of the oxygen feeder at a predetermined constant rate. The SHIRM bioreactor was turned on for 24 hours at 37°C until the culture reached the stationary phase at an oxidative potential of +100 mV.

The culture obtained after the first incubation can be referred to as the "P1 population" and is approximately 5 generations old. Frozen effluents from this batch were designated FFR-P1.1 and FFR-P1.2. 2.5 ml FFR-P1.1 was seeded into a new SHIRM reactor containing SBCM 7.1/V _0.2 . Subculturing continued until a 6th FFR-P6 subculture was obtained and the corresponding FFR-P was maintained at the stage of each intermediate subculture. At this stage, the cysteine concentration decreased to 3 mM and the cystine concentration increased to 5 mM while the oxidation potential was 600 mV relative to Ag/AgCl. At this stage, re-plating was carried out up to the 27th subculture, keeping all conditions constant, while fresh medium was used for each subculture. After the 27th subculture, seeding continued until the 30th subculture, as shown in Figure 5.

EXAMPLE 2

Selection of an improved strain adapted to an oxidizing environment

Various FFR-Ps from Example 1 were analyzed to select a strain adapted to an oxidizing environment and oxygen that could be used for further production.

The analysis is based on comparative metagenomic, metabolic characteristics, growth kinetics, stability and tolerance.

Comparative metabolic characterization is carried out by fatty acid profiling, more specifically butyrate production on various prebiotics such as inulin and resistant starch.

Comparative characterization of growth kinetics, including biomass yields and growth rates, is carried out in normal culture media with or without bile salts and prebiotics.

Comparative characterization of stability and tolerance is carried out in the presence of simulated gastric fluid/simulated intestinal fluid with or without enzymes and exposure to ambient air for 30 minutes.

Simulated gastric fluid test solution (TS) is prepared according to USP guidelines by dissolving 2.0 g sodium chloride and 3.2 g purified pepsin (derived from porcine gastric mucosa, with an activity of 800 to 2500 units per mg protein) in 7.0 ml of hydrochloric acid and water up to 1000 ml. This test solution has a pH of approximately 1.2.

The test solution (TS) of simulated intestinal fluid according to USP is prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate in 250 ml of water, and then adding 77 ml of 0.2 N sodium hydroxide and 500 ml of water. 10.0 g pankeratin is added and the resulting solution is adjusted to pH 6.8 ± 0.1 using 0.2 N sodium hydroxide or 0.2 N hydrochloric acid and finally diluted to 1000 ml.

Microbial fuel cell and flavin-dependent reduction of 5,5'-dithiobis-2-nitrobenzoate were used to evaluate cytochrome expression in FFR-M and FFR-P cell populations, while diaphorase activity was tested using nitrosine tetrazolium salt.

Choose the most adapted strain.

EXAMPLE 3

Additional example of adaptation of an anaerobic microorganism to an oxidized environment

All steps of Example 1 were followed up to the stage of obtaining a culture after the first incubation, designated as "population P1", which is approximately 5 generations old. Frozen effluents from this batch were designated FFR-P1.1 and FFR-P1.2. 2.5 ml FFR-P1.1 was seeded into a new SHIRM reactor containing SBCM 7.1/V _0.2 . Subcultures continued to the 6th subculture, obtaining FFR-P6, and the corresponding FFR-P was maintained at the stage of each intermediate subculture. At this stage, the cysteine concentration decreased to 3 mM and the cystine concentration increased to 5 mM while the oxidation potential was 600 mV relative to Ag/AgCl.

But in this example 3, compared to example 1, this re-plating step was carried out up to the 10th subculture in total, keeping all conditions constant, with fresh medium used for each subculture, as shown in figure 8.

EXAMPLE 4

Selection of improved strain from example 3

Various FFR-Ps from Example 3 were analyzed to select a strain adapted to an oxidizing environment and oxygen that could be used for further production.

The selection of an adapted strain is based on the following steps:

a. At each stage shown in figure 8, an aliquot of 100 μl was taken.

b. The "step a" sample was serially diluted (up to 10 ³ ) in air-saturated phosphate-buffered saline (PBS) as shown in Figure 9 and incubated in airtight vials at ambient temperature.

c. The vessels were placed in an anaerobic chamber and a 50 μl aliquot was seeded with YCFAG medium (Table 4).

d. The plates were incubated anaerobically for 24-72 hours.

e. After incubation, the number of viable cells was visually assessed.

f. Depending on the morphological type of the colonies, any variant obtained after improvement was selected and purified by the classical pure culture method.

g. Adapted strains were checked for purity by Gram staining.

h. The five adapted strains selected in step f were designated TCS1, LTCS, STCS, OCS-1 and OCS-2.

i. Details of the isolated adapted strains, including the relevant conditions and isolation steps, are presented in Table 2.

j. Preliminary identification of adapted strains is based on Gram stain, metabolic phenotyping (short chain fatty acid profiles) and quantitative PCR (qPCR) using F. prausnitzii -specific primers.

k. Strain identification was confirmed by 16S ribosomal DNA sequencing.

l. Oxygen tolerance and stability of the adapted strains were evaluated as shown in Figure 10. The respective adapted strains were anaerobically cultured in YCFAG medium for 12-14 hours and serially diluted to 10^one, ten², ten³, ten^four and/or 10⁵. 100 µl or 50 µl of serially diluted cultures were plated on plates with YCFAG medium in two parallels. One set of plates was incubated under anaerobic conditions and served as a control, while the other set was incubated aerobically for 20 minutes. Within 20 minutes of exposure to ambient air, oxygen completely diffuses into the agar plate, changing its color to pink. The redox indicator, resazurin dye, which is colorless in the reduced state, turns pink upon oxidation. This ensures complete diffusion of oxygen into the culture medium in the dish. After the respective treatments shown in Figure 10, the test and control cups were incubated under anaerobic conditions for 48-72 hours and the number of viable cells was visually assessed.

m. After strain confirmation by 16S ribosomal DNA sequencing, adapted strains of F. prausnitzii TCS1, OCS-1 and OCS-2 were selected based on their oxygen tolerance and deposited in accordance with the Budapest Treaty at the DSMZ (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under accession numbers DSM 32380, DSM 32378 and DSM 32379, respectively.

n. The oxygen tolerance of the F. prausnitzii type strain, the parental strain, and the adapted strains are shown in Table 3.

Table 2: Identified adapted strains in different cycles

Strain Code Full title Selection conditions one TCS1 Translucent homogeneous colonies 6th cycle 3.5/V _0.6 (1:100) 2 LTCS Large translucent homogeneous colonies 10th cycle 3.5/V _0.6 (1:1000) 3 STCS Small translucent uniform colonies 10th cycle 3.5/V _0.6 (1:1000) four OCS-1 Opaque homogeneous colonies 10th cycle 3.5/V _0.6 (1:10) 5 OCS-2 Opaque homogeneous colonies 10th cycle 3.5/V _0.6 (1:10)

Table 3: Stability profiles of adapted strains after exposure to air

Treatment Anaerobic Aerobic cfu/ml Faecalibacterium prausnitzii A2-165 (DSM 17677) 1.0E+0.8 0 Faecalibacterium prausnitzii FBT-22 (DSM 32186) 1.5E+0.7 0 Faecalibacterium prausnitzii TCS1 (DSM 32380) 7.0E+0.7 1.0E+03 Faecalibacterium prausnitzii OCS1 (DSM 32378) 6.5E+0.7 1.0E+05 Faecalibacterium prausnitzii OCS2 (DSM 32379) 7.5E+0.7 7.0E+05

Table 4: YCFAG medium for F. prausnitzii

g/l Kaziton ten Joint autoclaving yeast extract 2.5 NaCl 0.9 _{K2HPO4 _} 0.45 _{KH2PO4 _} 0.45 _NaHCO3 four CaCl _2, 2H ₂ O 0.12 MgSO _4, 7H ₂ O 0.09 Glucose 4.5 sodium acetate 2.7 cysteine one ammonium sulfate 1.32 Resazurin 0.001 Gemin 0.01 vitamins µg/l Addition after autoclaving Biotin (B7) ten Sterilization by filtration Cobalamin (B12) ten PABA thirty Folic acid fifty Pyridoxamine 150 Riboflavin (B2) fifty Thiamine (B1) fifty SCFA mm Addition after autoclaving propionate 9 Sterilization by filtration isobutyrate one isovalerate 2 valerate 3

The adjusted final pH is approximately 7.2.

EXAMPLE 5

Genetic characterization of improved strains

The improved strains obtained from each subculture in Example 1 are characterized by next generation sequencing (NGS). FFR-M and all FFR-P cultures are subjected to NGS for a thorough genomic analysis to identify any possible mutations occurring in the progeny of the improved/adapted FFR-P. Differences between FFR-M and FFR-P strains are also examined at the transcriptomic level by RNA sequencing.

EXAMPLE 6

Increased production of anaerobic microorganism

Production conditions will include the optimized conditions achieved in Example 1. The microorganism culture conditions of Example 2 will be the same as in Example 1, but will include only one stage with fixed oxidation conditions including voltage, a cysteine/cystine couple, and oxygen influx.

The optimized fermentation conditions are as follows: a membrane septum having a mass transfer coefficient (K _Om ) of oxygen of 1.3×10 ^-4 cm/s and an oxygen diffusion rate into the anode chamber of approximately 0.2 nmol⋅ml ^-1 min ^-1 , applied voltage 0.6 V vs. Ag/AgCl, cysteine 3 mM, cystine 5 mM, and an initial redox potential of the SBCM growth medium of approximately -188 mV.

The fermentation step is carried out in a 150 liter electrically insulated vessel made of fluoropolymer based plastic material with a triple chamber configuration. Figure 4c. It was sown using the preparation as in example 2 above.

The fermentation cell suspension is separated using an Alfa Laval continuous centrifuge and mixed with standard cryoprotectants as is known in the art. The washing step is carried out to avoid freezing point depression during lyophilization.

In lyophilization, the cell suspension is poured onto each dish in a lyophilizer. The Faecalibacterium prausnitzii cell suspension has a dry matter content of 18% and is lyophilized for four to five days.

Otherwise, the fermentation and lyophilization of the microorganism is carried out as is known in the art.

Claims (23)

1. A method for adapting anaerobic bacterial strains and selecting oxygen-tolerant anaerobic bacterial strains that exhibit an increased level of tolerance to ambient air compared to parental or parental strains that are not oxygen-adapted, comprising the steps (a) cultivating said bacterial strains with stepwise dual induction of oxidative stress by (i) applied voltage and oxygen diffusion, and (ii) stepwise decrease in antioxidant concentration combined with stepwise increase in concentration of oxidized form of the antioxidant, thereby changing the ratio of antioxidant/oxidized form concentrations antioxidant and redox adjustment to obtain adapted anaerobic bacterial strains with increased oxygen tolerance, where the concentration of dissolved oxygen is maintained at a sub-lethal concentration at which some, but not all, bacterial strains die; and (b) selection of oxygen-adapted anaerobic bacterial strains. 2. The method of claim 1 wherein the antioxidant/oxidized form of the antioxidant is selected from the group consisting of cysteine/cystine, glutathione/oxidized glutathione, ascorbic acid/dehydroascorbate, dithiothreitol/oxidized dithiothreitol, and gallic acid/oxidized gallic acid. 3. The method according to claim 1 or 2, characterized in that said method comprises a stepwise decrease in the concentration of the antioxidant in combination with a stepwise increase in the concentration of the oxidized form of the antioxidant, a stepwise increase in the applied voltage, and a stepwise increase in oxygen supply. 4. The method according to any one of claims 1 to 3, wherein the initial condition for said method is a culture medium containing (a) an oxygen concentration of zero to less than 50 μM, (b) an applied voltage of zero, (c) a high an antioxidant concentration, or (d) a low concentration of the oxidized form of the antioxidant, or any combination of (a)-(d), wherein said initial conditions result in a redox potential close to that of the intestinal lumen. 5. The method of claim 4 wherein the initial conditions are selected from one or more or all of 8 mM cysteine, 0 mM cystine, 0.1 V applied voltage, and zero or low oxygen supply. 6. The method of claim 5, wherein the stepwise change is continued until one or more, or all, of: a minimum cysteine concentration of 3 mM, a maximum cystine concentration of 5 mM, a maximum applied voltage of 0.6 V, and a high oxygen influx are achieved. 7. The method according to any one of claims 1 to 5, wherein the applied voltage does not exceed 0.6 V and/or the cysteine concentration is at least 3 mM and/or the cystine concentration does not exceed 5 mM and/or the concentration of dissolved oxygen in the medium for cultivation is maintained at the level of sublethal concentration. 8. The method according to any one of claims 1 to 7, wherein, after completion of the step change, the method comprises one or more additional steps of cultivating said bacterial strains under the end conditions of applied voltage, oxygen diffusion, and the ratio of antioxidant/oxidized form of antioxidant concentrations achieved after the step changes . 9. The method according to claim 8, further comprising the steps listed in claim 1, in which additional step changes are made. 10. The method of claim 1, wherein each step of increasing in (a)(i) and decreasing and increasing in (a)(ii) comprises reseeding the anaerobic bacterial strains into a new culture medium, resulting in adapted anaerobic bacterial strains. 11. The method of claim 10, wherein said passages include said incremental changes in culture conditions (i) and (ii). 12. The method according to any one of claims 1 to 11, wherein the bacterial strains are Faecalibacterium prausnitzii . 13. The method according to any one of claims 1 to 12, characterized in that said method includes an additional step in which the adapted strains are screened for oxygen tolerance, butyrate production and growth kinetics to select the most adapted strain with maintained metabolic characteristics. 14. A method for producing oxygen-adapted anaerobic bacterial strains, comprising adapting anaerobic bacterial strains and selecting more oxygen-resistant anaerobic bacterial strains that exhibit an increased level of tolerance to ambient air compared to parental or parental strains that are not oxygen-adapted, by the method according to any of claims 1-13 and further culturing bacterial strains using a combination of oxidative potential/applied voltage constant, oxygen diffusion, antioxidant concentration, and oxidized form concentration of the antioxidant that have been optimized to produce oxygen-adapted bacterial strains, with the dissolved oxygen concentration in the culture medium is maintained at a sub-lethal concentration, thus enabling selective pressure and obtaining a high yield of oxygen-adapted bacterial strains that exhibit increased increased level of tolerance to the ambient air compared to the original or parental strains that are not adapted to oxygen. 15. The method of claim 14, wherein said culture conditions include 3 mM cysteine, 5 mM cystine, an applied voltage of 0.6 V, and an oxygen influx of 0.2 nmol ml ^-1 min ^-1 . 16. The method according to claim 14 or 15, wherein said bacterial strain is Faecalibacterium prausnitzii . 17. The use of an oxygen-adapted bacterial strain obtained by the method of any one of claims 1 to 13 as a probiotic, wherein said bacterial strain exhibits an increased level of tolerance to ambient air compared to the original or parental strain that is not oxygen-adapted. 18. Use according to claim 17, wherein said bacterial strain is a strainFaecalibacterium prausnitzii, selected from the group consisting of DSM 32380, DSM 32378 and DSM 32379. 19. Oxygen adapted strainFaecalibacterium prausnitzii, which exhibits an increased level of tolerance to ambient air compared to the original or parental strain, which is not adapted to oxygen, for use as a probiotic, characterized in that said bacterial strain is a strainFaecalibacterium prausnitzii DSM 32380. 20. Oxygen adapted strainFaecalibacterium prausnitzii, which exhibits an increased level of tolerance to ambient air compared to the original or parental strain, which is not adapted to oxygen, for use as a probiotic, characterized in that said bacterial strain is a strainFaecalibacterium prausnitzii DSM 32378. 21. Oxygen adapted strainFaecalibacterium prausnitzii, which exhibits an increased level of tolerance to ambient air compared to the original or parental strain, which is not adapted to oxygen, for use as a probiotic, characterized in that said bacterial strain is a strainFaecalibacterium prausnitzii DSM 32379.

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