KIT FOR CONTROLLED EMISSION OF BIOLUMINESCENCE AND USES

Abstract

The invention relates to a kit for the controlled emission of bioluminescence from a bioluminescent bacterium in the presence of oxygen, said kit comprising, on the one hand, a lyophilized culture of said bioluminescent bacterium, and on the other hand, a medium for the culture of said bioluminescent bacterium, said substrate being in liquid or semi-liquid form, and the applications of such a kit.

Description

Description Title of the invention: KIT FOR CONTROLLED EMISSIONS OF BIOLUMINESCENCE AND USES

[0001] — The invention relates to an application of bioluminescence by the development of a kit for the controlled emission of bioluminescence from a bacterium, and its uses lisations.

[0002] — Bioluminescence is a phenomenon of light production (luminescence) natural by living organisms based on two molecules, a substrate the lu- ciferin and an enzyme, luciferase. Luciferases are oxygenases that oxidize the luciferin using molecular oxygen leading to the formation of a molecule having electrons in an excited singlet state and whose return to the ground state releases energy in the form of photons. In some organisms, luciferin and the luciferases are combined into a single protein, the photoprotein, a protein on which molecular oxygen binds to it, and the phenomenon occurs as soon as this The photoprotein-oxygen complex is placed in the presence of a cofactor such as ions calcium or magnesium. The invention covers these two major mechanisms and In general, any bioluminescence mechanism involving a luciferin and a luciferase.

[0003] Numerous uses of bioluminescence are known in biology, in par- specific for marking purposes.

[0004] — In the present text, the invention will be described with reference to bioluminescence of bacterial origin, the scope of the invention is not limited to this and extends to This phenomenon occurs regardless of the microorganism of origin. For example, it can It could be a dinoflagellate microorganism; it could also be a ge- organism netically modified.

[0005] — Bacterial bioluminescence is interesting in that the emitted light is visible to the human eye, especially at night. Bioluminescence is generally measured using a luminometer which expresses the result in URLs (relative units of light) or a photomultiplier (photons / second). We consider that the notion of visible light is located from 10E+7 photons / second.

[0006] — Thus, even outside the field of biology, its applications have already been the subject of numerous studies, yet without any real commercial success.

[0007] By way of example, document WO 97 / 29319A1 describes an article that can be a decorative object, textile, cosmetic product, food or beverage, Equipped with a system capable of generating bioluminescence, this system includes a mixture of at least one luciferin and one luciferase, particularly of bacterial origin which, upon contact with molecular oxygen, triggers bioluminescence. It can be implemented in a variety of ways. The mixture can be encapsulated in a capsule, for the preparation of bioluminescent drinks among other things, in which the capsule is dissolved, the bioluminescence being generated by the contact of the mixture with the oxygen contained in the drink; for its application in decoration, either luciferin or luciferase can be fixed on a solid support, which is then brought into contact, for example by vaporization, with the other compound, the bioluminescence manifesting in the air. For all these applications, the generated light is strictly dependent on the concentration of the reactants, and bioluminescence ceases upon their depletion or oxygen deprivation. It can be sustained for a few minutes but decays rapidly, with a half-life of approximately 5 hours. While the range of potential applications for bioluminescence is extremely broad, this duration is too short to generate significant industrial interest, and to date, no fields are known where it is used on a large scale. Document WO2018 / 087504A1 describes a system for the bioluminescent illumination of a decorative element, a building facade, or a piece of street furniture. The system's principle is based on the continuous or semi-continuous circulation of a culture of bioluminescent microorganisms within the area to be illuminated. This system consists of a largely transparent circuit for the circulation of the culture, which is connected to a reactor in which the culture grows and which supplies the circuit with culture. The reactor is itself supplied by a source of culture medium and a source of biomass. The system is equipped with a means for removing excess biomass and various sensors, including those for monitoring the state of the culture within the reactor.While this system can maintain long periods of luminescence, up to two weeks, it requires complex technology, making it difficult to implement on an industrial scale and easily compromised by even the slightest parameter affecting the culture of microorganisms. According to the invention, a solution is provided that overcomes the drawbacks of existing solutions, specifically both the strict dependence between the consumption of a reagent and the emission of light, and the complex implementation of the phenomenon. The invention stemmed from the discovery that a freeze-dried culture of bioluminescent bacteria could be reactivated by a suitable medium, allowing, on the one hand, a freeze-dried product to become the reservoir of reagents necessary for the manifestation of bioluminescence, and on the other hand, for the bioluminescence to be initiated at a time chosen by the user. Thus, the invention relates to a kit for the controlled emission of bioluminescence from a bioluminescent microorganism in the presence of oxygen, said kit comprising, on the one hand, a lyophilized product of a culture of said bioluminescent microorganism, and on the other hand, a medium allowing the culture of said bioluminescent microorganism, said medium being in liquid or semi-liquid form. According to one embodiment, the invention relates to a kit for the controlled emission of bioluminescence from a bioluminescent bacterium in the presence of oxygen, said kit comprising, on the one hand, a lyophilized product of a culture of said bioluminescent bacterium, and on the other hand, a medium allowing the culture of said bioluminescent bacteria, said medium being in liquid or semi-liquid form. By simply contacting the lyophilized powder with the medium in the presence of oxygen, the process is initiated and, depending primarily on the bioluminescent bacterium, the composition of the medium, and the environment in which the kit is used, the light is immediately visible and intensifies rapidly. Thanks to this design, once visible, the light is maintained for several tens of hours, expanding the possibilities of bioluminescence. Before describing the invention further, the definition of terms used in this text is given below. The term "kit" according to the invention refers to an assembly of at least two parts, namely a lyophilized culture of a bioluminescent bacterium on the one hand, and a medium for culturing said bacterium on the other, which are essentially sealed off from each other and are intended to be brought into contact to produce bioluminescence in the presence of oxygen. Any arrangement of said part relative to the other may be considered, but arrangements allowing easy contact between the contents are preferred. The medium for culturing said bioluminescent bacterium, according to the invention, is understood to be a medium comprising at least the nutrient substrates and a suitable buffer system necessary and sufficient for the growth of bacterial cells. Said nutrient substrates include at least one or more carbon sources, one or more nitrogen sources, one or more sulfur sources, one or more phosphorus sources, mineral nutrients, and optionally vitamins. This definition is within the general knowledge of a person skilled in the art, who can prepare or obtain commercially such a medium suitable for the bacterium to be cultured. This medium may also contain any other ingredient that promotes bacterial growth. The expression of bioluminescence, namely the emitted light, depends on bacterial growth; the greater the number of bacteria, the higher the light intensity.It is also the responsibility of a person skilled in the art to determine and select optional ingredients that can supplement the medium, particularly to contribute to optimal bioluminescence expression. This definition... applies in the same way to a medium allowing the culture of a microorganism other than a bacterium. It should be noted that bioluminescence is an environmentally sensitive phenomenon and may not occur in the absence of the required conditions, such as temperature, acidity, and oxygen content, even if the emitting bacterium remains alive. Thus, regardless of the bacterium and its resistance, bioluminescence is temperature-sensitive and generally does not occur at temperatures exceeding 35°C. Similarly, it may be affected in an environment with a pH below or above the range of 6-8.5. These constraints are known to those skilled in the art and are provided for illustrative purposes only; the invention is, of course, not limited to the expression of bioluminescence under these conditions. The invention is described in more detail below, all the features indicated below being considered alone or in any combination of two or more. The freeze-dried culture of a bioluminescent bacterium, constituting one part of a kit of the invention, can be prepared from a bacterial culture carried out under conventional conditions, which may optionally be modified specifically to promote cell growth. The higher the cell concentration in the freeze-dried culture, the more immediate and intense the light emission will be upon contact of the two parts of the kit. According to one embodiment of the invention, this culture is carried out in a medium identical to that constituting the other part of the kit. All bioluminescent bacteria are potential candidates for a kit of the invention. Due to their bioluminescence yield, a bioluminescent bacterium from any of the following genera is preferred: Aliivibrio, Enterovibrio, Photobacterium, Photodesmus, and Vibrio. Bacteria exhibiting both strong bioluminescence and good resistance to freeze-drying are also preferred. According to an advantageous variant, a marine bacterium of the species Photobacterium phosphoreum or Photobacterium kishitanii, or a bacterium of the species Vibrio gigantis or Vibrio huyengisis, is selected. It was previously stated that bioluminescence depends on physical parameters of the environment; it is primarily dependent on bacterial growth, and promoting growth contributes to better luminescence. Depending on the intended applications, certain species will be preferred due to their more cost-effective cultivation or their resistance to certain conditions, particularly freeze-drying. For example, Photobacterium phosphoreum and Photobacterium kishitanii are advantageously chosen because they continue to emit light at relatively low temperatures (4°C). The ideal acidity of the medium is relatively Common to all bioluminescent bacteria studied to date, this pH fluctuates between 6 and 8.5. Oxygen availability, essential for the bioluminescence reaction, is another crucial factor. The affinity of the bacterial bioluminescence system for oxygen also varies among species. For example, those of the genus Photobacterium have demonstrated greater resistance, and sometimes even superior bioluminescence, at low oxygen concentrations compared to other bioluminescent bacterial species. Even more advantageously, it is a bacterium of the strain Photobacterium phosphoreum ANT-2200. This strain was deposited under the Budapest Treaty with the CNCM depositary authority on February 24, 2021 under CNCM deposit number I-5659. It can be envisaged that the lyophilized product is a lyophilized product of a culture of several bioluminescent bacteria or is derived from a lyophilization of a mixture of cultures of different bacteria or a mixture of lyophilized products of cultures of different bacteria. The lyophilized product can be obtained by any lyophilization technique well known to those skilled in the art. According to a conventional method, the bacterial culture, preferably in the exponential phase, is placed in a lyophilization medium and subjected to lyophilization in any commercially available lyophilizer. The bacterial cell concentration of the culture placed in the lyophilization medium preferably ranges from 0.6 to 0.8 ODU (optical density units) at 600 nm. For optimal preservation of the viability of the cells undergoing lyophilization, the culture advantageously contains one or more cryoprotectants, such as glycerol or sucrose. In another embodiment of the invention, the aforementioned bacterial culture is directly lyophilized without prior suspension in a lyophilization medium and may, in this case, not require the use of any cryoprotectant. According to a preferred embodiment of the invention, the bioluminescent bacterium is a bacterium of the species Photobacterium phosphoreum, and the lyophilized powder is prepared by lyophilization of a culture medium of said bacterium into which a cryoprotectant has been introduced. This cryoprotectant is advantageously sucrose at a concentration of 12% (w / w) in said culture medium. The medium for culturing said bioluminescent bacterium, constituting the other part of a kit of the invention, is formulated in such a way that it meets all the conditions necessary for the culture, in the sense of growth or development, of said luminescent bacterium present in the lyophilized powder. To this end, it comprises at least one or more carbon sources, one or more nitrogen sources, and one or sources of sulfur, one or more sources of phosphorus, mineral nutrients and possibly vitamins. These requirements are generally met by the presence of the following ingredients: one or more peptones: a peptone is a protein hydrolysate, usually in powder form, rich in amino acids and short peptides which will provide carbon and nitrogen, as well as sulfur with methionine and cysteine; it also contains many coenzymes, as well as mineral nutrients, notably providing phosphorus; Yeast extract(s): This is a yeast lysate which is rich in carbohydrates and contains amino acids, nitrogenous bases, enzymatic cofactors, as well as mineral nutrients providing phosphorus among others. The medium also includes a buffer system capable of maintaining the culture's pH at an optimal value or as close as possible to it. This system is water-based, and if the bacteria are of marine origin, it is seawater-based. A medium in a kit of the invention may include any other complementary ingredient contributing to increasing the expression of bioluminescence, its intensity and / or its duration. It may thus include vitamins, such as Balch vitamins [WE Balch et al. (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43:260-296]. It can be liquid, or it can be semi-liquid in the sense that it tends towards a more viscous texture, provided that this does not impede the diffusion of the lyophilized powder when the two parts of the kit are brought into contact. To this end, it includes a gelling agent; if this is agar, the medium can contain up to 15 g / L of agar. Advantageously, the environment that allows cultivation promotes the development of the culture up to the exponential phase. Another advantage of the invention lies in its wide range of implementation options, provided that, until the desired triggering of the phenomenon, the parts are sealed from each other and a source of molecular oxygen is accessible. Thus, in one embodiment, the lyophilized powder and the culture medium for the luminescent bacteria are each contained in a flexible or rigid reservoir made of any material impermeable to solids and liquids, for example, plastic, transparent or opaque, these reservoirs being connected directly or not. In another embodiment, the lyophilized powder and the medium are each contained in a flexible or rigid reservoir made of any plastic material impermeable to solids and liquids, but permeable to gases, including oxygen or an oxygen source such as air.In another embodiment of a kit of the invention, the lyophilized powder and the medium are each in a flexible or rigid container, impermeable to solids, liquids, and gases, made of any suitable material, for example, glass or plastic. In this case, oxygen or an oxygen source must be available. To produce bioluminescence, for example, the atmosphere in one or both reservoirs may be sufficient; it can also be supplied by a third reservoir included in the kit. The reservoirs may or may not be connected. Thus, the kit may consist of a container comprising the reservoirs for the kit components, said container having a separable section for contacting the contents of the two components. In a particular version, the separable section of said container contains oxygen or an oxygen source. Regardless of the variant, the material is preferably transparent, or at the very least it lets through all or almost all of the emitted light. Of course, the lyophilized powder and the medium can be in compartments made of different materials, and any combination is possible. It can also be constructed in various configurations; for example, at least two parts of the kit can be connected end-to-end, or one can surround all or part of the other. Depending on the intended application, it may be preferable to use several kits of the invention, identical or different, rather than a single kit whose container volume would be too large. Thus, the invention also relates to multiple kits, identical or different, as defined above, said kits being, for example, enclosed in a solid package, such as a box. By way of example, the kit may be in the form of a capsule with a flexible or rigid shell, consisting of at least two separate parts, one containing the freeze-dried culture of at least one luminescent bacterium, the other containing a medium for culturing said bacterium or bacteria, the two parts at least being separated by a partition that can be broken to combine the two parts. This partition may be breakable, its rupture then being caused by an appropriate treatment such as applying pressure to the capsule or, preferably, to a group of capsules.In another variant, it can be soluble, its contact with a solvent specifically disintegrating it or disintegrating both the partition and the capsule's outer layer. The partition can also be broken by a change in temperature or by exposure to light. According to another embodiment, a capsule can be refilled with lyophilized powder and culture medium, respectively. The invention relates to any application of a kit or multiple kits described above. Thus, it provides an article equipped with one or more kits, said article being a decorative object, an event product, a lighting accessory, a signage device, or a part of a building structure. The invention further relates to a method for illuminating a support, according to which a kit of the invention and a support are available, the lyophilized product and the medium are brought into contact, and then the resulting mixture is applied to said support for its illumination. Since bioluminescence is observed when the kit components are brought into contact in the presence of oxygen, it is possible to consider that oxygen may be present either from the moment the media are brought into contact, or only at the time the mixture is applied to the support. Thus, according to the method of the invention, the lyophilized powder and the medium are brought into contact in the presence or absence of oxygen. According to an alternative, the lyophilized powder and the medium are brought into contact in the absence of oxygen, and the mixture is applied to the support in the presence of oxygen. The application can be carried out using any appropriate technique, such as spraying, misting, or atomization. It can be adapted to the surface to be treated and / or the printing technique; thus, for treating large and / or complex surfaces or for stenciling, spraying is the preferred method. For this purpose, the user can use a paint sprayer, a spray gun, an airbrush, etc. The invention is illustrated in the following examples which highlight its advantages. Example 1: Preparation of a lyophilized culture of a bacterium of the strain Photobacterium phosphoreum ANT-2200 A culture of P. phosphoreum ANT-2200 is carried out at 19°C in a ZoBell-type liquid medium comprising, per 1000 mL of medium: 5 mg of peptone 3 mg of yeast extract 3 mL of glycerol 750 mL of seawater 250 mL of H₂O and whose pH is adjusted to 7.5. 25 mL of this culture at the end of the exponential phase (determined by an optical density OD of approximately 0.7 and a bioluminescence of approximately 10E+7URL) are collected and centrifuged for 20 minutes at 8000 rpm. The resulting pellet is resuspended in 200 µL of supernatant and then transferred to a 1.5 mL Eppendorf tube. 200 JL of a 24% (w / w) sucrose solution is then added. The sample is subjected to rapid freezing in liquid nitrogen and then stored at -80°C for 3 days. The sample is then lyophilized for 24 hours. The resulting lyophilized sample is exposed to a nitrogen stream for 1 minute and then stored at room temperature. Example 2: Effectiveness of a kit according to the invention The effectiveness of a kit of the invention is evaluated using a lyophilized product of Example 1, which constitutes part of the kit, and a ZoBell-type liquid medium as described in Example 1 constitutes the other part of the kit according to the protocols indicated below. the essay: At ty, the entire lyophilization tube is resuspended in 5 ml of ZoBell biopeptone medium (in a 50 ml bottle) then 250 jl of this suspension are spread on a Petri dish also containing ZoBell biopeptone. After 3 hours, the liquid culture begins to glow. After 14 hours, the culture glows intensely. For up to almost 3 days, the bioluminescence is measured regularly, and the following values ​​are determined: at 5:30 PM, 3 10E7 at ty. 24h 3 10E7, at t,, 42h 2.8 10E7, at to, 49h 2.8 10E7: a clear brightness is still observed, at ty, 67h 2.4 10E7 2nd attempt: At to, the entire contents of a lyophilization tube are added to 10 ml of ZoBell biopeptone medium (in a 50 ml bottle) and then 250 jl of this suspension are spread onto a Petri dish also containing ZoBell biopeptone. For up to 3 days, bioluminescence is regularly measured; the following values ​​are measured: at to, 3pm, 1.16 10E7, at to, 20h 2.98 10E7, at to, 24h 2.05 10E7, at to, 39h 1.3 10E7, at to, 45h 1.3 10E7, to, 76h 7 10E6. As long as the bioluminescence value is greater than 10E7, then it is perfectly visible to the naked eye. These two tests demonstrate the performance of a kit of the invention, at least 3 days after contacting the lyophilized culture with a culture medium according to the invention, in the presence of ambient air, even at low cell concentrations of the lyophilized.

Claims

Demands

1. Kit for the controlled emission of bioluminescence from a bio- bacterium luminescent in the presence of oxygen, said kit comprising, on the one hand, a lyophilized product of a culture of said bioluminescent bacteria nescente, and on the other hand, a medium for the culture of said bioluminescent bacteria nescent, said substrate being in liquid or semi-liquid form.

2. Kit according to claim 1, characterized in that the bioluminescent bacterium nescente is chosen from among the species Photobacterium phosphoreum, Photobacterium kishitanii, Vibrio gigantis and Vibrio huyengisis.

3. Kit according to claim 2, characterized in that the bioluminescent bacterium nescente is a bacterium of the Photobacterium phosphoreum strain ANT-2200, which was filed with the CNCM on February 24, 2021 under CNCM filing number I-5659.

4. Kit according to any one of claims 1 to 3, characterized in that that the lyophilized powder and the culture medium for the luminescent bacteria are each in a reservoir, flexible or rigid, made of any material Waterproof against solids and liquids, for example, plastic, transparent or not, the said tanks being connected directly or not.

5. Kit according to claim 4, characterized in that the tanks are in gas-tight material.

6. Kit according to any one of claims 1 to 5, characterized in that that it is in the form of a container comprising the reservoirs, said container having a breakable part for bringing the two so-called biological environments.

7. Kit according to claim 6, characterized in that the breakable part of said container contains oxygen or an oxygen source.

8. Multiplicity of kits according to any one of claims 1 to 7, identical or different, and enclosed in sturdy packaging, such as than a box.

9. Article supplied with one or more kits according to any of the re- sales 1 to 7, the said item being a decorative object, a product event lighting, a lighting accessory, a signage device, a part of the structure of a building.

10. A method for illuminating a support, characterized in that it has a kit according to any one of claims 1 to 8 and of a support and in The lyophilized powder is brought into contact with the medium, and then the resulting mixture on said support.

11. The method according to claim 10, characterized in that one puts in contact the lyophilized product and the medium in the presence of oxygen.

12. A method according to claim 10 or 11, characterized in that one puts in Contact the lyophilized powder and the medium in the absence of oxygen and apply the mixture on said support in the presence of oxygen.

13. A method according to any one of claims 10 to 13, characterized in that the mixture is applied to said support by spraying, ne- bulization or atomization.