PREPARATION OF MICROORGANISMS FOR THE FERMENTATION OF VEGETABLES, EXTRACTS OBTAINED AND THEIR USES

Abstract

The invention relates to a preparation of microorganisms comprising, in a sweetened aqueous nutrient medium, a consortium of microorganisms comprising at least one lactic acid bacterium, for example of the genus lactobacillus or pediococcus, at least one yeast, for example of the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, and at least one acetic acid bacterium, for example of the genus acetobacter, gluconobacter, or komagataeibacter. It also relates to a method for producing a plant extract rich in active compounds, by fermentation in the presence of said consortium of a substrate which may be derived from a whole plant, a part of a plant, or a plant co-product. Also claimed is the use of these extracts for the manufacture of pharmaceutical and cosmetic products, perfumes, food supplements or plant treatment products.

Description

Title of the invention: PREPARATION OF MICROORGANISMS FOR THE FERMENTATION OF VEGETABLES, EXTRACTS OBTAINED AND THEIR USES

[0001] The present invention belongs to the field of the extraction of active compounds of plant origin and their valorization. It relates to a composition of microorganisms, a process for fermenting plants using this composition and the use of the plant extracts obtained in various applications.

[0002] Plants have been used for thousands of years for their beneficial properties. They contain many active compounds such as polysaccharides, oligosaccharides, proteins, lipids, but also vitamins, minerals and many other substances with particular biological activities. The use of these substances involves extracting them from the plant tissues containing them and making them available in a form suitable for the intended use, particularly in the fields of health, cosmetics, perfumery, food, or even for plant health.

[0003] Among the classic methods, there are solvent extraction processes during which the plant material rich in the compound of interest is immersed in a liquid having an affinity with it. Organic solvent extraction techniques, long predominant, are today criticized due to the pollution induced by these solvents which generate ecotoxic waste whose recycling is difficult and whose disposal is not very respectful of the environment. In addition, these processes are very energy-intensive and often require special confinement conditions (supercritical CO2, subcritical water) making their implementation complex. An alternative may be oil phase extraction using solvents of natural origin, such as vegetable oils. However, this technique only allows the extraction of lipophilic compounds.Aqueous phase extraction has low yields, unless specific processing conditions are implemented to improve extraction rates: high temperature, addition of co-solvents or additives. The extracts obtained are fragile and are subject to microbial contamination which must be controlled to ensure sufficient preservation by strict control of operating conditions (in particular pH, ionic strength, pressure, temperature, sterility).

[0004] Another approach is to use plants as raw material for transformations orchestrated by microorganisms. Many processes involving selected or modified microorganisms are described in literature and are used in industrial settings. The most widespread are based on fermentation, known for millennia as a food preservation process and defined as the transformation of carbon substrates in the absence of oxygen. Fermentation relies on the substrate's endogenous microflora or on added microorganisms, generally chosen for their fermentative abilities or resistance to environmental conditions. In these processes, a type of microorganism transforms a given substrate, for a single, specific objective. For example, we can cite the processes of winemaking (grapes - yeasts - wine production) or beer brewing (barley malt - yeasts - beer production).

[0005] Furthermore, biotechnological processes using microorganisms on suitable synthetic nutrient media are used to produce compounds of interest such as vitamin B12, citric acid, lactic acid or even hyaluronic acid. These fermentations, or rather transformations carried out by microorganisms, are demanding in terms of implementation: temperature, pH, ionic strength, specific nutrient medium, synthesis inputs, strictly controlled atmosphere (gas, pressure), aseptic conditions (absence of contaminants). They are followed by purification steps that are costly, both in terms of energy and purification solvents and waste treatment. In all cases, here again, these processes concern a substrate - microorganism - single objective triptych.

[0006] However, it is known that in nature, microorganisms organize themselves within communities that interact, which allows them to biodegrade organic matter, even in unfavorable environments. These communities, called consortia (consortium in the singular), develop synergistic behaviors thanks to close metabolic couplings. Therefore, it has become interesting to design combinations of appropriately chosen microorganisms and to use them for agricultural purposes. Document WO2019 / 133923 describes, for example, compositions comprising a synthetic consortium of several microorganisms, and their use in foliar application to stimulate plant growth, or during silage operations. Another promising avenue is the production of hydrogen by degradation of biomass.Work carried out to optimize the production of bio-hydrogen has highlighted the existence of a physical interaction and exchanges of cytoplasmic molecules between the microbial cells of a consortium selected from a natural soil consortium.

[0007] The present invention aims to take advantage of these cooperative abilities between microorganisms, to extract different types of compounds from plants. A fermentation process using such a consortium must make it possible to degrade the biomass and extract or make available compounds present in a plant substrate, without resorting to synthetic inputs and without rejection of polluting waste. These operating conditions, which are also energy-efficient, meet the objective of developing so-called eco-responsible technologies.

[0008] An aim of the present invention is thus to propose a consortium of microorganisms, defined and stable, capable of reacting with a plant substrate, in an aqueous medium. Another aim of the invention is to propose a consortium capable of fermenting and transforming all kinds of plant substrates, to extract and make accessible the compounds they contain. Yet another aim of the invention is to carry out an efficient extraction of the active compounds from different plants, including compounds that are difficult to access by other techniques, with a high yield. Yet another aim of the invention is to obtain extracts rich in various active compounds, usable as such, or for the subsequent preparation of products for pharmaceutical, cosmetic, food, phytosanitary, or other purposes.

[0009] The present invention is based on a novel approach, according to which a single microbial consortium works with a wide variety of plants and with any type of plant or plant-derived substrate, liquid or solid, to extract numerous compounds available in the form of aqueous extracts or powder, easy to store and to use in all kinds of applications. Statement of the invention

[0010] A first subject of the invention thus relates to a preparation of microorganisms comprising a consortium of microorganisms in a sweetened aqueous nutrient medium, said consortium comprising i) at least one microorganism belonging to the group of lactic acid bacteria, ii) at least one microorganism belonging to the group of yeasts and iii) at least one microorganism belonging to the group of acetic acid bacteria.

[0011] The microorganisms making up the artificial consortium thus created according to the invention are chosen from three groups present in nature, due to their complementary activities. They are preferably non-genetically modified microorganisms.

[0012] Yeasts are aerobic-anaerobic eukaryotic cells that can be described as biological multi-reactors. They use carbon substrates as sources of carbon and energy. They are capable of metabolizing many sugars, with the exception of polysaccharides. In particular, they produce CO2 and alcohol, as well as many aromatic molecules.

[0013] Acetic acid bacteria consume simple sugars to produce, among other things, organic acids such as gluconic acid and vitamins. They also consume alcohol from the metabolism of yeasts and lactic acid bacteria, transforming it into acetic acid. In addition, they are capable of synthesizing a biofilm, which will play an important role in the stability of the consortium, as we will see later.

[0014] Lactic acid bacteria, when in a suitable medium, induce rapid acidification of the medium, with concomitant synthesis of bacteriocins which thus ensure their protection against pathogens and other Gram + microorganisms, such as Bacillus or others. However, they are demanding with regard to their development conditions and are not very resistant to acidification of the medium in free forms. It is known that the presence of yeasts contributes to their growth.

[0015] It is rare that under natural conditions, these three groups find conditions favorable to their growth at the same time and in the same place. The microbial consortium is therefore an artificial assembly of microorganisms, which must be able to coexist and collaborate to form a balanced, stable and reproducible consortium. This assembly is further designed to combine complementary activities. In the composition present, genera of microorganisms have been favored, within which particular species have been retained as more favorable to these multiple requirements.

[0016] For lactic acid bacteria, microorganisms of the order Lactobacillales of the Lactobacillaceae family of group 1 are advantageously used, in particular those belonging to the genera Lactobacillus and Pediococcus. For example, two food strains such as Lactobacillus plantarum and Lactobacillus acidophilus can be used, with a preference for Lactobacillus plantarum because it is commonly found on plants.

[0017] Yeasts of the order Saccharomycetales of the family Saccharomycetacea or of the order Schizosaccharomycetales of the family Schizosaccharomyce etaceae may be advantageously used, in particular those of the genera Saccharomyces, Schyzosaccharomyces or Torulaspora. For example, baker's yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii, brewer's yeasts Saccharomyces boulardii and Schyzosaccharomyces pombe may be chosen, alone or in combination.

[0018] For acetic acid bacteria, bacteria of the order Rhodospirillales of the Acetobacteraceae family are preferably used, with in particular the genera Acetobacter, Gluconobacter and Komagataeibacter (Gluconacetobacter). These acetic acid bacteria can originate from an unpasteurized cider or wine vinegar with an acidity level of 4% to 5%, advantageously from a commercial cider vinegar, labeled "Organic Agriculture" according to the definition of the European Union. Inoculation is therefore carried out by direct addition of an aliquot of vinegar to the composition. In vinegars, alongside the dominant acetic acid bacteria, there exists a complex natural indigenous flora, part of which will be able to coexist with the added microorganisms. Another part will perish during the establishment of the consortium, in the same way that the symbiotic development of all the microorganisms forming the consortium prevents the development of undesirable species, including pathogenic species.

[0019] This is why, according to a preferred characteristic, the preparation of microorganisms according to the invention comprises: - at least one lactic acid bacterium belonging to the genus lactobacillus or pediococcus, - at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, - at least one acetic acid bacterium belonging to the genus acetobacter, gluconobacter, or komagataeibacter.

[0020] According to a particularly preferred characteristic of the preparation of microorganisms which is the subject of the invention, the latter comprises: - at least one lactic acid bacterium chosen from Lactobacillus plantarum and Lactobacillus acidophilus, - at least one yeast chosen from Saccharomyces cerevisiae, Saccharomyces boulardii, Schyzosaccharomyces pombe and Torulaspora delbrueckii, - at least one acetic acid bacteria from cider vinegar or wine vinegar.

[0021] The culture medium also plays an important role. In the composition according to the invention, the medium is a sweet nutrient medium, i.e. rich in sugars that can be assimilated by the microorganisms present. These carbohydrates, generally mono- or disaccharides, can come from various sources. It can be a pure sugar, generally sucrose, glucose or fructose, or a transformed product or co-product, such as molasses. Mixed sources are possible although more complex to manage. Note that since sucrose can be assimilated by many microorganisms, and in particular by those of the consortium according to the invention, it will be preferred.

[0022] Originally, the aqueous phase of the nutrient medium is not pure water, but a plant extract. It can be an infusion, a decoction or a maceration or even a juice (raw liquid fraction obtained by pressing) of a plant or part of a plant, for example tea leaves, coffee beans, cocoa pods, or other. An infusion has the advantage that the plant is briefly subjected to high temperatures, ensuring suitable rehydration without degrading the extracted compounds. An infusion of tea (Camellia sinensis) has proven to be particularly suitable, with the added advantage of being very easy to use. Note that tea naturally has a complex indigenous flora, which will disappear in the presence of the consortium, or coexist in the composition without harmful consequences for its activity.

[0023] Thus, according to a preferred original characteristic of the invention, the nutrient medium comprises an aqueous tea extract and a source of assimilable sugars chosen from sucrose, glucose, fructose, molasses, or a mixture thereof. Typically, the nutrient medium may be an infusion of black tea prepared with 2 g / 1 to 50 g / 1 of tea leaves, with added sucrose at a concentration of between 20 g / 1 and 100 g / 1. Preferably, the tea is prepared with 5 g / 1 to 10 g / 1 of tea leaves, and sweetened with 50 g / 1 to 95 g / 1 of sucrose. The latter may be replaced by a mixture of glucose and fructose.

[0024] This medium, entirely plant-based and without synthetic input, ensures the vital comfort of each of the microorganisms. It offers each of them the possibility of satisfying its energy needs in the growth phase under well-defined batch culture conditions (discontinuous culture) in aerobic or anaerobic conditions. The microorganisms are introduced in dry or wet form into the sugary nutrient medium, with an inoculation rate commonly between 103 CFU / g and 105 CFU / g of medium. The microbial suspension obtained is incubated between 12°C and 45°C, preferably between 25°C and 30°C, for 6 to 21 days, at the end of which a composition of microorganisms with a total flora of between 105 CFU / g and 107 CFU / g is obtained. A microbiological count of the viable flora indicates that a balance of flora has been created in the medium. The consortium is visualized by the presence of a biofilm on the surface.The composition is maintained for as long as desired by subculturing on new nutrient medium. The composition can also be preserved by refrigerating it at 4°C or by freezing, for later use.

[0025] Thus, according to a preferred embodiment of the preparation of microorganisms which is the subject of the present invention, it is obtained by a process comprising: - the introduction of said microorganisms into said nutrient medium to obtain a microbial suspension, and - the incubation of the microbial suspension in an open reactor, at a temperature between 12°C and 45°C for 6 to 21 days, to obtain a stable consortium of said microorganisms in the form of a biofilm, comprising a total flora of 105 CFU / g to 107 CFU / g.

[0026] It has been observed that under the above-mentioned culture conditions, the microorganisms are able to grow together and form a balanced, stable and reproducible consortium. It is remarkable that the culture takes place in an open reactor, without it being necessary to take any special measures to ensure its asepsis with respect to exogenous contaminants, unlike conventional discontinuous culture methods. It can be hypothesized that the choice of microorganisms with varied and specific oxygen requirements favors the development of each population during a culture under the aforementioned conditions. Indeed, a distribution is created in the reactor which is dependent among other things on the O2 gradient of the medium and its redox potential, and also on the appearance of a biofilm produced by the microorganisms at the water-air interface. This biofilm advantageously protects the medium from environmental contaminants, whether chemical or biological. It materializes the cooperation of the three groups of microorganisms and contributes to the robustness of the process.

[0027] The consortium of the preparation of microorganisms according to the invention is capable of reacting with a plant substrate, in an aqueous medium. It is capable of directing the extractive fermentation of all kinds of plants, to make accessible, and possibly transform, a great diversity of the compounds that they contain.

[0028] Thus a second object of the present invention relates to a method for producing a plant extract, comprising the steps consisting of: a) - preparing a fermentation medium containing a plant substrate in aqueous phase, b) - inoculating said fermentation medium with a preparation of microorganisms described above in such a quantity that the inoculation rate is between 102 CFU / g and 104 CFU / g of fermentation medium, c) - incubate in an open reactor, at a temperature between 12°C and 45°C, for 2 to 20 days, d) - recover the supernatant, with or without the residual biomass, to obtain an extract of said plant rich in active compounds.

[0029] The extract obtained can therefore consist of the liquid supernatant alone or of the entire culture medium which can be harvested with the biomass and possibly undergo drying.

[0030] In a preferred embodiment of the process for producing a plant extract according to the invention, in step b), the fermentation medium is inoculated with a quantity of the preparation of microorganisms of between 0.5% and 5%, by mass relative to the mass of fermentation medium, and incubated at a temperature of between 25°C and 30°C, for 10 to 15 days. Preferably, the inoculation rate is between 0.8% and 3%.

[0031] As can be seen, even when inoculated at relatively low rates (i.e. between 102 CFU / g and 104 CFU / g), the consortium quickly colonizes the medium to induce fermentation. The process takes place in the presence of the plant, which becomes a continuous source of nutrients under the action of the biochemical tools of the different microorganisms. This allows it to be maintained in the culture medium for several days, or even a few weeks, without human intervention. Although fermentation normally takes place anaerobically, the process is conducted here in an open environment for gas exchange with the atmosphere, and without any particular constraints to ensure sterility. It is assumed that the conditions induced and tolerated by the consortium in the reactor prevent the development of pathogenic flora present on the substrate and in the environment, even under non-aseptic conditions.

[0032] The inoculated composition of microorganisms is capable of directing the extractive fermentation of all kinds of plants. The substrate can be chosen from higher plants of all types, whether flowering plants, aromatic plants, cereals, fodder plants, protein crops, oilseeds, or others, representing a wide variety of compounds that can be used and enhanced. It can also belong to lower plants, including ferns, mosses, fungi, lichens and algae. It can also be chosen from organisms found in phytoplankton, that is to say all plant organisms living in suspension in water, autotrophic with respect to carbon, which includes in particular microalgae and bacteria such as cyanobacteria (formerly "blue-green algae").This is why, according to a characteristic of the method according to the invention, the plant substrate is chosen from higher plants, lower plants or phytoplankton organisms.

[0033] As can be seen, the fermentation process which is the subject of the invention is highly versatile and has a general, even universal, character. Remarkably, the consortium is able to adapt to different types of substrates sensitive to putrefaction (cereals, fodder plants, algae, protein-rich plants, etc.), rich in minerals or aromatic compounds (rosemary, lavender, chamomile, ginger, turmeric, coffee, tea, cocoa, etc.), as well as to single-celled organisms such as spirulina. Whatever the substrate, we will use, as far as possible, sources of supply which comply with the standards of organic farming which refuses the use of synthetic chemical products.

[0034] According to particular embodiments of the invention, the plant substrate can be nettle (Urtica dioica), woad (Isatis tinctoria), Damask rose (Rosa damascena), Jujube (Ziziphus zizyphus), Ginkgo biloba, rosemary (Rosmarinus officinalis), lavender (Lavandula), chamomile (Roman chamomile Chamaemelum nobile or wild chamomile, German, matricaria, etc.), heather (notably the species Calluna vulgaris), ginger (Zingiber officinale), turmeric (Curcuma longa), coffee (Rubiaceae coffea), tea (Camellia sinensis), cocoa (Theobroma cacao), or even spirulina (Spirulina).

[0035] The plant substrate may include a whole plant or a part of a plant. It may further be fresh or dried, native or processed. The part of the plant chosen may be the one that is known in the state of the art as containing compounds of interest or having particular properties, such as turmeric root, jujube fruit, lavender flower, etc. Other parts of the plant can also be chosen, which may reveal their content of neglected or even ignored active compounds until today, but which can be extracted and used thanks to the synergistic action of the microbial consortium according to the invention. Plant co-products can also be used: the pastel leaves remaining after extraction of the pigments by decoction are a promising substrate as a source of interesting compounds for other applications. In addition, the plant substrate can be in a solid form or in a form with a predominant liquid component. Their implementation will be adapted accordingly.

[0036] According to a first embodiment of the invention, the plant substrate can be obtained in a solid form, that is to say in the form of fragments, ground material or powder, from a whole plant or part of a plant, or a solid co-product resulting from a treatment of said plant for other purposes and in other processes.

[0037] According to the invention, for the production of a plant extract suitable for a substrate in solid form, in step a), said substrate is mixed with water and then brought to a temperature of between 25°C and 90°C, and if necessary, sugar is added, before or after bringing to temperature, to bring the fermentation medium to a Brix level of between 2 and 10. The addition of sugar can be up to 100 g / l of fermentation medium, or be unnecessary when the substrate itself is sufficiently sweet.

[0038] According to a second embodiment of the invention, the plant substrate may be in a liquid or essentially liquid form, obtained in particular in the form of a juice, an infusion, a decoction or a maceration, from a plant or a part of a fresh or dried plant, or as a liquid co-product resulting from a treatment of said plant. This concerns fresh fruit juices, flower nectar, as well as all plants in the aqueous phase with or without heating. Fragments may remain dispersed in the liquid phase, such as fruit pulp, the leaves of an infusion, or spirulina cells. The liquid phase resulting from the extraction of the pastel after precipitation and decantation of the pastel pigment is a co-product which must be mentioned among others which are also of interest.

[0039] According to a method of producing a plant extract suitable for a substrate in essentially liquid form, in accordance with the invention, in step a), the plant substrate is heated to a temperature of between 25°C and 85°C, and if necessary, sugar is added before or after bringing the fermentation medium to a Brix level of between 2 and 10. The sugar addition can be up to 100 g / l of fermentation medium if necessary. For example, the liquid substrate with 50 g / l of sucrose added is heated to a temperature of around 70°C for 5 to 10 minutes. A fraction of dechlorinated water can be added to dilute the juices in certain cases that the person skilled in the art will be able to identify.

[0040] In cases where in step a), the addition of sugar is required to bring the fermentation medium to the desired level, it is possible to proceed in different ways, and in particular, the fermentation medium can be sweetened by adding a source of assimilable sugars chosen from sucrose, glucose, fructose, molasses, honey, or a mixture thereof. It should be noted that the honey can make its own contribution to the properties of the extract obtained. For example, certain honeys particularly rich in minerals and polyphenols, such as heather or chestnut honey, can express their antiseptic power or other beneficial effects reinforcing those conferred by the active ingredients originating from the plant substrate.

[0041] The plant extract obtained is stable. It is remarkable that no putrefaction phenomenon is observed during the extraction-fermentation process, whereas plant substrates are generally loaded with microbial flora causing their degradation, as is particularly the case with nettle. It can be used immediately or preserved by a known technique. In any case, it can undergo different operations before being used or stored, preferably between 4°C and 18°C.

[0042] At the end of the extraction-fermentation process, one can focus on the supernatant in which extracted compounds are dissolved, in which case the process for producing a plant extract according to the invention can comprise: - a filtration step of the fermented extract to eliminate plant residues, - a sterilizing filtration step to eliminate microbial flora, commonly carried out on a 0.2 pm filter.

[0043] These filtrations will be omitted if the biomass is preserved as is the case when one seeks to recover active ingredients remaining in the biomass, but made bioavailable by fermentation and the action of the enzymatic equipment of the microorganisms of the consortium. It is specified that in the present description, the expression "plant extract" designates the product resulting from the extraction process by fermentation, with or without filtration, and both with and without the plant and microbial biomass of the fermentation medium.

[0044] Whether the plant extract is a solute or a mass, it will be necessary to stop fermentation and other undesirable processes in order to stabilize the product obtained. Any known means may be used, for example pasteurization or sterilization by heating, microbiological decontamination by a technique used in the food industry (gamma rays, hydrostatic pressure, etc.). Aqueous extracts may be lyophilized, solid extracts may be dried, for example by placing them in an oven between 30°C and 45°C, and they may be ground more or less finely depending on the intended use.

[0045] The method according to the invention can thus comprise, after step d), one or more of the following operations: filtration of the fermented extract to eliminate plant residues, sterilizing filtration to eliminate microbial flora, microbiological decontamination, freeze-drying, drying in an oven, grinding. The person skilled in the art will be able to choose the appropriate techniques depending on the initial state of the extract and the desired form for its conservation and subsequent use.

[0046] The process just described saves a lot of time and reduces operations involving operators. It meets current requirements in terms of eco-responsibility: no synthetic input, low-energy and asepsis-free open system culture conditions, and operating autonomy during the process. It is industrializable and economically profitable. It is emphasized that it provides access to compounds that are rarely extracted by conventional solvent methods or even by targeted enzymatic methods. This ability is attributed to the fact that the consortium at work offers a wide variety of biochemical tools, some of which are capable of breaking down supramolecular structures of the plant, simplifying macro-compounds to make them soluble and thus releasing them. Therefore, the process can be described as an extraction-fermentation-transformation process (all in one).

[0047] A plant extract containing active compounds, obtained by a process as described above, is also the subject of the present invention. As explained above, this extract can be produced from a multitude of plants, the plant being able to be chosen from higher plants, lower plants or phytoplankton organisms. In particular, a plant extract produced from a substrate chosen from flowering plants, aromatic plants, cereals, fodder plants, protein crops, oilseeds, and in particular nettle, woad, Damask rose, jujube, ginkgo biloba, rosemary, lavender, chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina may be concerned.

[0048] As explained above, the plant extracts obtained using the composition of microorganisms by the extraction method according to the invention are characterized by a great diversity of compounds obtained, and as a corollary a great variety of biological activities, physicochemical properties and organoleptic properties, in particular olfactory properties, with in certain cases, interesting synergies. They are therefore particularly suitable for use in compositions developed with a view to implementing these activities. They can in particular be formulated in compositions, which are also the subject of the present invention, such as creams, lotions, gels or other forms, intended for various uses. This is the case of a composition comprising an extract fermented plant according to the invention, or capable of being obtained by a process according to the invention, and at least one compound chosen from: an excipient, a natural additive, a synthetic additive, a thickener, a stabilizer, an emulsifier, an adjuvant, or a mixture thereof.

[0049] Whatever the specific formulation, the plant extracts according to the invention are ingredients which can be used in numerous and varied applications, in particular for the manufacture of a pharmaceutical product, in particular a dermatological product, a cosmetic product, a perfume, a food supplement or a plant treatment product.

[0050] According to one embodiment, a plant extract which is the subject of the invention can be used for the manufacture of a product for cosmetic or therapeutic purposes for cutaneous application, in particular a cream. This cream can, for example, comprise one or more fermented plant extracts such as a nettle extract, a ginkgo biloba extract, a pastel extract, or others.

[0051] A plant extract according to the invention can also be used for the manufacture of a perfume comprising one or more fermented plant extracts, for example a jujube extract, a tea extract, or any other odorous extract that the expert imagines assembling.

[0052] According to another embodiment, a plant extract according to the invention can be used for the manufacture of a food supplement, for example comprising a fermented spirulina extract rich in free phycocyanin, obtained after filtration of the fermentation medium, or obtained by dehydration and grinding in the presence of the fermentation medium.

[0053] According to yet another embodiment, a plant extract according to the invention can be used for the manufacture of a product for phytosanitary use. For example, a nettle extract and / or a tea extract, intended for an application such as the prevention or treatment of leaf lesions, water regulation, or the stimulation of plant growth.

[0054] The present invention will be better understood, and relevant details will appear, in the light of the description which will be given of different variant embodiments, in relation to the appended figures, in which:

[0055] [Fig. 1] represents the evolution of the iron content in a nettle extract according to the invention.

[0056] [Fig.2] represents the evolution of the total acidity in the same nettle extract.

[0057] [Fig.3] shows the rate of living fibroblasts at different concentrations of nettle extract El.

[0058] [Fig.4] shows the rate of living keratinocytes at different concentrations of the extract El.

[0059] [Fig.5] shows the influence of this nettle extract on different activities.

[0060] [Fig.6] shows the rate of live fibroblasts at different concentrations of the Ginkgo biloba E3 extract according to the invention.

[0061] [Fig.7] shows the rate of living keratinocytes at different concentrations of the same Ginkgo biloba extract.

[0062] [Fig.8] presents the influence of this extract E3 on different activities.

[0063] [Fig.9] gives the absorption spectra of Turmeric extracts according to the invention.

[0064] [Fig. 10] shows the rate of living fibroblasts at different concentrations of extract E6 (pastel leaves from decoction) according to the invention.

[0065] [Fig. 11] shows the rate of live keratinocytes at different concentrations of the same extract.

[0066] [Fig. 12] shows the rate of living fibroblasts at different concentrations of the E7 extract (co-produced juice of pastel) according to the invention.

[0067] [Fig. 13] shows the rate of live keratinocytes at different concentrations of the same E7 extract.

[0068] [Fig. 14] shows the rate of living fibroblasts at different concentrations of the E8 extract (native pastel leaves) according to the invention.

[0069] [Fig. 15] shows the rate of live keratinocytes at different concentrations of the same E8 extract.

[0070] [Fig. 16] shows the influence of extract E6 on different activities.

[0071] [Fig. 17] shows the influence of extract E7 on different activities.

[0072] [Fig. 18] shows the influence of extract E8 on different activities.

[0073] [Fig. 19] gives the absorption spectra of a spirulina fermentation medium according to the invention.

[0074] [Fig.20] represents the effect of extracts according to the invention on plant cells.

[0075] EXAMPLE 1: Preparation of a composition of microorganisms

[0076] The selected microorganisms are introduced in dry or wet form into sweetened black tea constituting the nutrient medium. The tea is prepared from 2 g / l to 50 g / l of dry leaves infused in water at a temperature of 70°C to 90°C for 10 min to 60 min. Commonly, 5 g / l to 10 g / l of tea leaves infused for 15 min at 80°C are used. The infusion can be used as is with the leaves, or more conveniently the aqueous phase is filtered. The sucrose is then dissolved in the aqueous phase at a concentration of between 20 g / l and 100 g / l, generally 50 g / l. The sucrose can be replaced by a mixture of glucose and fructose. The inoculation rate is between 103 CFU / g and 106 CFU / g of medium depending on the groups of microorganisms. The acetic acid bacteria are provided by a quantity of unpasteurized vinegar such that the pH of the nutrient medium remains within a range of 4 to 5.Microorganisms grow easily under defined conditions. The microbial suspension is incubated between 25°C and 30°C for 6 to 21 days, under non-aseptic conditions in an open reactor, advantageously cylindrical in shape, so that the system is dynamic and self-regulation occurs. After about two weeks, a biofilm forms, indicating the creation of the consortium. The consortium is maintained in the nutrient medium by respecting a consortium / medium mass ratio ranging from 50 / 50 to 20 / 80. After about 3 weeks, the pH and Brix degree have decreased, indicating that the sugars are consumed. Subculture on fresh nutrient medium is then carried out using conventional techniques known in the state of the art.

[0077] Compositions of microorganisms have been developed according to this procedure.

[0078] Composition Cl: Composition Cl is prepared according to Example 1 using two dietary strains of lactic acid bacteria, Lactobacillus plantarum and Lactobacillus acidophilus, two strains of baker's yeast, Saccharomyces cerevisiae and Torulaspora delbrueckii and unpasteurized wine vinegar with 5% acidity, in an infusion of black tea prepared with 8 g / l of leaves infused for 15 minutes at 85°C then filtered, and with the addition of 50 g / l of sucrose. The consortium biofilm forms after 15 days.

[0079] Composition C2: Composition C2 is prepared according to Example 1 using two food strains of lactic acid bacteria, Lactobacillus plantarum and Lactobacillus acidophilus, two strains of baker's yeast, Saccharomyces cerevisiae and Torulaspora delbrueckii and unpasteurized cider vinegar with 5% acidity. The ferments are introduced into an infusion prepared with 10 g / l of tea leaves infused for 20 minutes at 80°C then filtered, and to which 70 g / l of sucrose are added. The consortium biofilm forms from 15 days, although a little less significant than in composition CL

[0080] Formation of the consortium: A microbiological count of the viable flora was carried out after 5 days of culture. It indicates the presence of a total flora of between 106 CFU / g and 107 CFU / g, with a similar representation of each microbial group. For comparison, each microorganism of composition Cia was cultured individually under the same conditions: sweet tea culture medium, an inoculation rate close to 106 CFU / g, reactors under non-aseptic conditions, temperature of 25°C to 30°C. After 5 days, the lactobacilli did not survive and the yeasts were contaminated. The test with vinegar alone showed no change: neither contamination nor growth of acetic acid bacteria. Furthermore, sweetened black tea identical to the nutrient medium of composition C1 was incubated under the same conditions. It showed contamination by molds after 5 days.None of the tests showed biofilm formation. EXAMPLE 2: Preparation of plant extracts

[0081] Extract El Nettle powder (Urtica dioica) is mixed with dechlorinated water heated to 80°C at a rate of 50 g / kg of medium. Sucrose is then added at a rate of 50 g / kg of medium and left to cool. Composition C2 prepared according to Example 1 is added at a rate of 1% and the whole is incubated at 28°C for 11 days. Nettle extract E1 is obtained, with the nettle powder residue. The solid and liquid fractions can then be separated by filtration, centrifugation or other technique known per se, depending on the intended applications.

[0082] Extract E2 Nettle powder (Urtica dioica) is mixed with dechlorinated water heated to 80°C at a rate of 50 g / kg of medium. The medium is then sweetened at a rate of 50 g / kg of medium and left to cool. Composition C2 prepared according to Example 1 is added in a proportion of 2% to the unfiltered aqueous nettle suspension. The nettle mixture with the powder residue is incubated for 11 days. The test supernatant is removed and then subjected to a first filtration to remove plant residues and a second sterilizing filtration on 0.2 µm to remove the ferments. Extract E2 is obtained.

[0083] Extracts of rosemary (Rosmarinus officinales), Damask rose (Rosa damascena) and jujube leaves (Ziziphus zizyphus) were prepared according to an identical protocol.

[0084] Extract E3 Ginkgo biloba in the form of 50 g / kg of leaf powder is placed in dechlorinated water heated to 80°C. Sucrose is added at a rate of 50 g / kg. Composition C2 prepared according to Example 1 is added to the aqueous suspension of Ginkgo at a rate of 2%. The whole is incubated for 13 days at 28°C. The test supernatant is removed and then filtered to remove plant residues and sterile filtered to remove ferments.

[0085] Extract E4 Turmeric (Curcuma longa, root powder) is placed at 200 g / kg in sugar water at 50 g / kg heated to 80°C for 15 min. The medium is stirred and allowed to cool before adding the ferments of composition C2 prepared according to example 1, at 2%. The test is incubated for 15 days at 28°C. At the end of the extraction / fermentation, the fermented powder is recovered and dried. A sample is taken and taken up in water at 200 g / kg to characterize it. It is compared to non-fermented Turmeric powder taken up in water at 200 g / kg.

[0086] Visual observation of the two samples shows a clear difference in behavior: the supernatant of the fermented Turmeric extract is yellow in color. and cloudy, indicating the solubilization of compounds such as curcumin or other colored molecules, whereas Turmeric in water is translucent with significant sediment after decantation, indicating very few solubilized compounds.

[0087] Extract E5 A fermented extract of black tea (Camellia sinensis) was prepared by infusing 8 g / l of leaves in water at 80°C, then sucrose was added at a rate of 50 g / l. After cooling to room temperature, the composition Cl prepared in Example 1 is added at a rate of 2%. The whole is incubated at 25°C for 14 days. The liquid phase acidified to a rate of 2% (total acidity in acetic acid equivalent) is taken and then filtered through a 0.2 μm filter.

[0088] Extract E6 The extraction of the pastel blue pigment from the plant of the same name (Isatis tinctoria) requires processing a large quantity of leaves in a decoction in water. The pigment yield is low since 1 tonne of fresh leaves extracts 1 kg of pigment. The extraction process targeted at pastel therefore generates large quantities of co-products, which may contain compounds of interest for other applications. Obtaining the pigment uses enzymatic tools from the endogenous flora of the plant, indicating its fermentable nature. However, uncontrolled fermentation of Isatis leaves gives rise to the development of putrid flora releasing pestilential odors when spreading or storing this waste.We were interested in three co-products, namely the fresh leaves after decoction in water (extract E6), the liquid phase from the extraction of pastel obtained after precipitation and decantation of the pastel pigment (extract E7) and the juice from the decoction of the fresh leaves (extract E8).

[0089] Isatis leaves having undergone a first extraction by decoction were used to prepare the fermented extract E6. To do this, 8.5 kg of whole wet leaves (containing approximately 15% dry matter) are first subjected to a decoction in 20 kg of sugar water at 50 g / kg at 60°C. Then, 30 kg of cold dechlorinated water are added to complete and cool the preparation. When it reaches 30°C, composition C2 prepared according to Example 1 is added, at a level of 2%. The whole is incubated for 10 days at 28°C. The fermentation supernatant is removed and then filtered to remove plant residues, then sterilely on 0.2 μm to remove the ferments.

[0090] The extract obtained has a fresh and vegetal odor, characteristic of Isatis, while the initial microbial load of the wet leaves was high since it was greater than 3.107 CFU / g. The controlled and orchestrated fermentation by the consortium of microorganisms of composition C2 therefore made it possible to overcome the endogenous flora of the Isatis leaves. The minerals released during fermentation were measured by inductively coupled plasma spectrometry (ICP-OES) or by mass spectrometry inductively coupled microscopy (ICP-MS). The released calcium rate was 218.0 mg / kg, magnesium 31.4 mg / kg and iron 9.15 mg / kg. These mineral concentrations are significant and demonstrate the benefit of fermenting the wet leaves from the decoction for their decomplexation and release.

[0091] Extract E7 A fermented extract of a juice remaining after precipitation and decantation of the pastel pigment was prepared. The objective is to valorize this co-product which is currently discarded, by treating it by fermentation. The juice was heated to 80°C and, at this temperature, sucrose is added at a rate of 50 g / l and maintained at this temperature for 15 min in order to reduce the endogenous microbial load which amounts to 5.107 CFU / g. When the medium is cooled, composition C2 is added at a rate of 2% to the medium and the whole is incubated at 28°C for 10 days. The initial pH of the medium is 5.6 which is favorable to the development of the consortium. After 10 days, the fermentation is stopped by sterilizing filtration. The extract obtained is brown in color with a pleasant characteristic vegetal odor.

[0092] Extract E8 The juice obtained by decoction of native Isatis leaves at 200 g / l in water at 70°C before precipitation and decantation of the pastel pigment was fermented. To do this, the juice is heated for 15 minutes at 80°C, then sucrose is added at a rate of 50 g / l. The sweet juice is kept at this temperature for 15 minutes. After cooling, composition C2 is added at a rate of 2% to the medium and the whole is incubated at 28°C for 10 days. The initial pH of the medium is 4.5 which is conducive to the development of the consortium. After 10 days, the fermentation is stopped by sterilizing filtration.

[0093] The extract obtained is brown-green in color with a characteristic plant odor, and foaming. The minerals released during fermentation were measured by inductively coupled plasma spectrometry (ICP-OES) or by inductively coupled mass spectrometry (ICP-MS). The released calcium level is 321.0 mg / kg, that of magnesium 63.0 mg / kg and that of iron 15.2 mg / kg. These mineral concentrations are significant and interesting for cosmetic applications. The foaming power of the E8 extract can be used for mild cleaning products. It is also noted that the E6 extract obtained from leaves from pastel production made it possible to access quantities of minerals ranging from 50% to 70% of those obtained from native leaves (E8 extract). The process according to the invention shows that a recovery of pastel co-products should be considered.

[0094] Extract 9 Spirulina is a cyanobacterium used for human health due to its richness in micronutrients (minerals, vitamins, amino acids, pigments, acids essential fats). It is also known for its phycocyanin content, a blue pigment with numerous biological properties (antioxidant, anti-inflammatory, hepatoprotective, etc.). Extracting it currently requires complex processes involving multiple steps, solvents or synthetic adjuvants, or energy-intensive processes (ultrasound, microwaves, vortex, freezing / thawing). Spirulina extraction-fermentation has been carried out to release the phycocyanin and increase its shelf life in wet form.

[0095] Spirulina in the form of dry powder at 20 g / l is placed in dechlorinated water heated to 80°C. Sucrose is added at a rate of 50 g / kg. Composition Cl prepared according to Example 1 is added to the aqueous suspension of spirulina at a rate of 2%. The test is incubated for 6 days at 28°C.

[0096] Extract E10 Heather or common heather is known for its calming, antiseptic, and antioxidant properties due to the presence of tannins and polyphenols such as quercetin. Commercial preparations are alcoholic extracts that can cause side effects on health and, during preparation, on the environment. Extraction by fermentation in a completely aqueous medium offers a solution to this problem. It is also suggested to sweeten the fermentation medium with honey, although the acidity of heather and honey, combined with their antiseptic properties, create fermentation conditions unfavorable to the success of this extraction.

[0097] The fermentation of heather flowers (calluna vulgaris) was carried out in the presence of chestnut honey. The bud flowers are introduced at 10 g / l in water and infused for 30 min at 80°C-90°C. The infusion is filtered and when its temperature is lower than 45°C, chestnut honey is added at a rate of 70 g / l. When the temperature is lower than 30°C, the medium is inoculated with 20% of composition C2. After 12 days of fermentation at 26°C, the sugars are consumed and the rate of acid formed is close to 2.5%, which indicates, with the presence of a biofilm, that the consortium has developed. The supernatant is filtered through 0.2 μm to ensure its microbiological stabilization. It is found that the consortium according to the invention perfectly ensured extractive fermentation, despite difficult conditions.

[0098] EXAMPLE 3: Extraction of iron from a nettle extract

[0099] Since nettle is rich in iron, tests were carried out concerning its extraction by the process which is the subject of the invention. The fermentation medium with residue (noted ORT-AR) leading to the nettle extract E1 was compared with two other media prepared as follows: - Fermentation is carried out in a similar manner to that of El, but filtration of the medium is carried out before the addition of composition C2 to eliminate nettle residues (the fermentation medium is noted ORT-AR). - A control fermentation without inoculation of ferment is carried out by replacing composition C2 with water (the fermentation medium is noted ORT-SSF).

[0100] Samples of the supernatant from the three media were taken during fermentation at D0, D3, D6 and D12 and the iron was measured by colorimetry with potassium permanganate. The results obtained are presented in [Fig.l]. The samples with ORT-AR residues showed an increasing level of extracted iron over time, whereas this level did not change in the medium without ORT-SR nettle powder. This attests to the continuous extraction of iron from the plant residues during fermentation, the maximum extraction rate being reached from the 6th day by the action of the added ferments. Indeed, iron extraction was also visible in the samples without ORT-SSF ferment, but it was slower since it only reached its maximum after 12 days.On the other hand, ORT-SSF samples show a significant proliferation of native nettle flora, leading to the development of a strong, unpleasant odor, as well as a blackening of the product, signs of its degradation. On the contrary, ORT-AR samples are dark green with a strong plant odor. Total acidity was also measured by colorimetric assay with sodium hydroxide in the presence of phenolphthalein. It is expressed in % of acetic acid equivalents, as shown in [Fig.2]. The sugar level in degrees Brix was recorded on D0 and D6.

[0101] The difference in acidity between the three media is marked from the 6th day and continues until the 11th day. In the ORT-SSF test, the acidity stabilizes from the 8th day, which indicates that acid production has stopped, thus indicating the absence of fermentation strictly speaking, while the sugars are not completely consumed (on D6, the sugar is measured at 4.6 °Bx for an initial value of 4.9). On the other hand, in the presence of composition C2, the acidity increases regularly, with concomitant consumption of sugar (for the ORT-AR samples, the sugar is at 2.9 °Bx on D6, compared to 5.3 on D0; for the ORT-SR samples, the sugar is at 2.7 °Bx on D6, compared to 5.1 on D0). The production of acid with consumption of sugar during fermentation is characteristic of the establishment of the consortium of composition C2 in fermentation media.

[0102] These results show that the extraction / fermentation treatment according to the invention simultaneously made it possible to rapidly release the iron contained in the nettle and to prevent wild microbial development, protecting both the liquid extract and the residual biomass of the plant thus treated.

[0103] EXAMPLE 4: Biological properties of a nettle extract

[0104] Cytotoxicity The nettle extract E2 obtained in Example 2 was tested for its in vitro cytotoxicity on two cell types: normal human epidermal keratinocytes, representative of the epidermis, and normal human dermal fibroblasts, representative of the dermis. To do this, the extract is previously diluted in cascade in a culture medium adapted to each cell type in concentrations between 0.01% and 10%. The diluted extracts are placed in contact with the cells for 24 hours. During the last 3 hours, the colored reagent WST1 (Roche) is introduced into the medium. The optical density at 450 nm (yellow color) is proportional to the number of living cells. The results given in [Fig. 3] (fibroblasts) and [Fig.4] (keratinocytes), show that the E2 extract does not manifest any cytotoxicity compared to the control without extract, on both cell types, when it is present at a content between 0.01% and 1% in vitro, which is compatible with cutaneous applications.

[0105] Cellular activities The E2 extract was then tested on a genomic chip to identify the stimulated activities. The extract is pre-diluted in culture medium, at a non-toxic concentration of 0.01%, then incubated with normal human epidermal keratinocytes for 24 hours. Messenger RNA (mRNA) is extracted and then analyzed by RT-qPCR (Real Time quantitative Polymerase Chain Reaction). The results are shown in [Fig. 5]. They are expressed in %, positive in case of overexpression and negative in case of inhibition compared to gene expression in cells cultured under normal conditions.

[0106] An overexpression of 60% to more than 150% of the genes corresponding to the following activities is observed: fibrillins 1 and 2, collagen VII, occludin, syndecan, aquaporin, growth factor HBEGF (Heparin-Binding EGF-like Growth Factor), sirtuin, catalase and propiomellanocortin. The overexpression of these genes is interesting for cosmetic applications of the "anti-aging" type.

[0107] EXAMPLE 5: Biological properties of a Ginkgo biloba extract

[0108] Cytotoxicity The Ginkgo biloba E3 extract obtained in Example 2 was tested for its in vitro cytotoxicity on two normal human cell types: keratinocytes and fibroblasts, according to the procedure identical to Example 4. The results given in [Fig.6] (fibroblasts) and [Fig.7] (keratinocytes), show that the E3 extract does not manifest any cytotoxicity compared to the control without extract when it is present at a content of 0.01% to 1% in vitro, which is compatible with cutaneous applications.

[0109] Cellular activities The E3 extract was then tested on a genomic chip to identify the stimulated activities, after dilution in culture medium, at the non-toxic concentration of 0.01%, followed by incubation with normal human epidermal keratinocytes for 24 hours. Messenger RNAs (mRNAs) were extracted and analyzed as above. The results are shown in [Fig.8].

[0110] An overexpression of 60% to more than 100% of the genes corresponding to the aquaporin, filaggrin, involucrin and transglutaminase 1 activities and up to 320% for the peptidase-3 inhibitor are observed. On the other hand, the activity of the metalloprotease is inhibited. The changes in expression of these genes are interesting for cosmetic applications aimed at strengthening the barrier function of the skin.

[0111] EXAMPLE 6: Extraction of curcumin and derivatives of an extract of Curcuma longa

[0112] Turmeric is known to be naturally rich in curcuminoids, mainly curcumin. Extract E4 prepared in Example 2, whose visual observation suggests the solubilization of colored compounds, was analyzed. To do this, at the end of the extraction / fermentation process, the fermentation supernatant is separated from the residual turmeric powder, which is oven-dried at 45°C. The liquid phase (turmeric-F-juice) and the dried solid phase (turmeric-F-powder) are analyzed by spectrophotometry and compared to the native, unfermented turmeric taken up in water (turmeric-H2O). To this end, the fermented and native turmeric powders are taken up at 100 mg / ml and dilutions are made to be able to compare the samples. The samples are centrifuged and the supernatant is analyzed by visible spectrophotometry. The obtained spectra are presented in [Fig.9].

[0113] The spectra of the three samples show the presence of curcumin (absorbing between 400 nm and 430 nm) and other compounds from the process (absorbing between 450 nm and 500 nm). The curcumin content appears to be equivalent in the fermented turmeric supernatant (Curcuma-F-juice) and native turmeric (Curcuma-H2O) showing that fermentation did not degrade this compound. In addition, the fermented turmeric powder (Curcuma-F-powder) shows very marked curcumin peaks and metabolizing compounds, approximately four times greater than those of native turmeric. The extraction-fermentation process released 4 times more curcumin than a simple water extraction and therefore makes curcumin more accessible. These results show that curcumin is not degraded by fermentation and that the dried fermented powder is enriched in a bioavailable, water-extractable form of curcumin.

[0114] EXAMPLE 7: Biological properties of pastel extracts and pastel co-products

[0115] Isatis tinctoria, known for the production of pastel, contains other compounds which may have interesting properties for the cosmetic field. Tests were carried out on the three extracts E6, E7 and E8 of Example 2, after heating at 80°C for 30 min and sterilizing filtration.

[0116] Cytotoxicity Extracts E6, E7 and E8 were tested for their in vitro cytotoxicity on human keratinocytes and fibroblasts, according to the procedure described in Example 4, with extract concentrations between 0.01% and 5%. The results for extract E6 (fermented Isatis leaves, noted FIS AF) are given in [Fig. 10] (fibroblasts) and [Fig. 11] (keratinocytes). The results for extract E7 (fermented Isatis juice, noted JISAF) are given in [Fig. 12] (fibroblasts) and [Fig. 13] (keratinocytes). The results for extract E8 (untreated leaves, noted DECISA) are given in [Fig. 14] (fibroblasts) and [Fig. 15] (keratinocytes). They show that extract E6 does not show any cytotoxicity compared to the control when present at a content of 0.01% to 2.5% in vitro, extracts E7 and E8 being devoid of toxicity at a content of 0.01% to 0.5%. Cutaneous applications can therefore be envisaged.

[0117] Cellular activities Extracts E6, E7 and E8 were then tested on a genomic chip to identify stimulated activities. The extracts were diluted in culture medium suitable for keratinocytes, at a non-cytotoxic concentration of the extract, and then incubated with normal human epidermal keratinocytes for 24 hours. Messenger RNA (mRNA) was extracted and analyzed as in Example 4. The results are presented in Figures 16, 17 and 18 respectively for extracts E6, E7 and E8.

[0118] Extract E6 shows a marked overexpression of the genes corresponding to aquaporin 3 and involucrin, and a particularly high overexpression of the peptidase inhibitor 3 genes (approximately 350%). Concerning extract E7, several genes are overexpressed between 50% and 180% (aquaporin 3, Heparin-Binding EGF-like Growth Factor or HBEGF, involucrin, occludin, Serine Protease INhibitor Kazal-type 5 or SPINK5). The overexpression of the gene corresponding to the peptidase-3 inhibitor is particularly high at a level of approximately 450%. Concerning extract E8, several genes are overexpressed between 80% and 180%. The overexpression of the decorin and involucrin genes around 250% is particularly high.

[0119] It can be stated that the activities detected on the three Isatis-based extracts are interesting for cosmetic applications. It is further observed that the activities detected are different for each of the extracts.

[0120] EXAMPLE 8: Extraction of pigments from a spirulina extract

[0121] The E9 extract prepared in Example 2 was analyzed. On days JO, D3 and D6, an aliquot of spirulina was taken and centrifuged for spectrophotometric analysis. The spectrum obtained is shown in [Fig.16]. The spectrum on D6 indicates a greater absorption from 400 nm to 700 nm compared to D0 and presents the two characteristic peaks of spirulina, around 460 nm for carotenoids and around 600 nm for phycocyanin. The biofilm also contains phycocyanin.

[0122] On the other hand, it is known that spirulina is sensitive to microbial contamination in an aqueous medium due to its rich nutrient composition and its basic pH. It gives off a characteristic marine odor which can sometimes bother the consumer. A control sample of spirulina was incubated in water without ferments under the same conditions as above. After 1 day, the fermentation medium shows wild contamination, identified by the nauseating odor of putrefaction. On the contrary, in the test carried out with composition Cl, the unpleasant marine odor disappeared. It is therefore noted that the process according to the invention made it possible to avoid wild fermentation of the medium.

[0123] Thus, extraction / fermentation makes it possible to extract compounds of interest from spirulina and also makes it possible to sanitize spirulina in the aqueous phase. Therefore, it is possible to preserve it without adding preservatives. It can be used as a source of active ingredients, or. These operations can be carried out without inconvenience in the presence of the biomass of the consortium of microorganisms.

[0124] EXAMPLE 9: Care cream comprising a nettle extract

[0125] Nettle is generally used for its soothing properties on the skin. The activities present in extract E2 highlighted in example 3 are interesting for use in a care cream with a restructuring, firming, moisturizing effect. A formulation of care cream with fermented nettle extract was produced and showed that it is possible to incorporate it easily.

[0126] In 35 g of demineralized water, 0.5 g of xanthan gum is dissolved with vigorous stirring. Then, the emulsifying base (0.75 g of hydroxyethyl acrylate and sodium acryloyldimethyl taurate copolymer) is added with stirring. From 1 g to 5 g of fermented nettle extract E2 (1% to 10% of the final formula) are added to the previous mixture, which constitutes the aqueous phase. The oily phase is prepared by mixing coco caprylate (0.75 g), coconut butter (0.5 g) and jojoba oil (3.75 g). With vigorous stirring using a deflocculating paddle. The oily phase is added gently to the aqueous phase and the resulting emulsion is stirred gently for a few minutes. A preservative system is added, for example a mixture of benzyl alcohol, salicylic acid and sorbic acid. The resulting emulsion is adjusted to a pH between 5 and 5.5 with 1N sodium hydroxide and to a mass of 50 g with demineralized water.

[0127] The resulting cream is smooth, slightly shiny and moderately compact. The composition comprising 10% E2 extract is very slightly off-white with a slight characteristic nettle odor. It spreads easily on the skin, penetrates easily and leaves a silky and comfortable feel.

[0128] EXAMPLE 10: Care cream and gel comprising a Ginkgo biloba extract

[0129] Ginkgo biloba is generally used for its effects on blood microcirculation. The activities present in extract E3 highlighted in Example 4 are interesting for use on the skin. Extract E3 was incorporated without difficulty into a care cream and into a gel.

[0130] Care cream comprising an extract of Ginkgo biloba In 35 g of demineralized water, 0.5 g of xanthan gum is dissolved with vigorous stirring, then the emulsifying base (0.75 g of hydroxyethyl acrylate and sodium acryloyldimethyl taurate copolymer) is added with stirring. From 0.5 g to 5 g of E3 extract of Ginkgo biloba (1% to 10% of the final formula) is added to the previous mixture, which constitutes the aqueous phase. The oily phase prepared as in the previous example is gently added to the aqueous phase, as well as a preservation system. The emulsion thus obtained is adjusted to a pH between 5 and 5.5 with 1N sodium hydroxide and to a mass of 50 g with demineralized water.

[0131] The cream obtained, comprising 10% E3 extract, is quite compact and bright white. It does not give off the typical smell of the plant. It spreads easily and penetrates, leaving a silky feel and well-hydrated, clearer skin.

[0132] Aqueous gel comprising an extract of Ginkgo biloba In 45 g of demineralized water, 0.5 g of xanthan gum is added. The mixture is stirred vigorously until the gum is completely dissolved and thickened. From 0.5 g to 5 g of E3 extract of Ginkgo biloba (1% to 10% of the final formula) is added with moderate stirring. A preservative system, for example dehydroacetic acid and benzyl alcohol, is added. The resulting mixture is adjusted to a pH of 5 to 5.5 if necessary, and by mass to 50 g with demineralized water.

[0133] The gel obtained has a very gel-like texture which spreads well on the skin. EXAMPLE 11: Anti-itch lotion

[0134] The E10 extract based on heather and honey was tested for its effects on insect bites, particularly tiger mosquito bites. A few drops of pure extract were applied by light massage to the skin of patients who had spots in several places on their body, indicating an inflammatory reaction following mosquito bites. The patients reported no longer feeling any itching just 3 to 5 minutes after application. After 24 hours, the spots had all significantly regressed or even completely disappeared. This result shows that the E10 extract has properties marked anti-allergic and soothing properties, which can be used in an aqueous lotion. EXAMPLE 12: Perfume Creation

[0135] Perfumes in general are made up of an alcoholic part and an aqueous part in variable proportions. Plant extracts obtained according to the invention can advantageously replace the aqueous part of the perfumes, which makes it possible to enhance the odorous notes but also to bring a touch of care to the finished product. Since water and alcohol (ethanol) are miscible, the perfumes thus composed will be rather light and fresh. In addition, the addition of fermented plants in perfuming solutions has a very interesting antioxidant potential, making it possible to consider their use in perfumed care water formulations. The mixing of scents and the creation will be the work of the expert.

[0136] Different compositions were made by mixing fermented extracts with ethanol (EtOH). The nettle, rosemary, damask rose and jujube leaf extracts were fermented according to the protocol used for extract E2 of Example 2. The black tea extract E5 prepared according to Example 2 is also used.

[0137] Perfuming composition A A perfume composition A was created by mixing fermented extracts of rosemary (5%), Damask rose (54%), nettle (27%) and jujube leaves (14%). The dominant top note is that of rose, supported by rosemary, which is perceived later. To soften the whole, more subtle notes are provided by the other extracts.

[0138] Perfume compositions B1 and B2 Two compositions were created from a commercial eau de toilette with a very sweet and heavy peach scent. In composition B1, rosemary extract (very fragrant) was added at a rate of 20%. In composition B2, 20% fermented tea (not very fragrant but acetic) was added. The top note is different in the compositions, but suggests peach in a lighter and fresher way than in the initial eau de toilette. The new fragrances B1 and B2 obtained are invigorated by the presence of fermented extracts and the acidity brings volatility to the aromas, making them lighter and fresher.

[0139] EXAMPLE 13: Treatment of lesions on plants

[0140] The effects of a fermented tea extract are shown in an experiment on vine leaves. The vine leaf is injured in two places with a scalpel (1 and 2). On lesion 1, a drop of the black tea extract E5 described in example 2 was deposited, lesion 2 is left as is. A control is constituted by the deposit of a drop of the extract E5 on a part of the healthy leaf without lesion. After 2 days, a very clear evolution of the plant tissue of the injured area treated with the extract is observed. E5 on which the lesion area is delimited by diffusion of the E5 product and where the different lesions are no longer visible. On the contrary, they remain open and susceptible to the development of diseases on the untreated part. The healthy part of the leaf does not show any visible reaction of the leaf cells, attesting to the safety of the E5 extract. We can hypothesize an interaction of the polyphenols of the E5 extract with the structural polysaccharides of the vine leaf inducing a barrier effect. EXAMPLE 14: Action on plant stomata

[0141] Other effects of a tea extract according to the invention have been observed on plant cells and more particularly on the stomata, true sensory receptor organs responsible for the exchanges of the plant with its environment.

[0142] A few drops of the E5 tea extract were applied to the leaves of various plants: iris (Iris germanica), chives (Allium schoenoprasum), porcelain plant (Graptopetalum paraguayen.se). The cells observed under an optical microscope (magnification x 600) have widely open stomata. Thus, the application of the fermented tea extract induces a mechanical response of the plant cells and facilitates cellular exchanges with their environment, as a blue light would do on a plant, thus accelerating cell growth. This E5 extract promotes the receptivity of the tissue while maintaining a healthy terrain.

[0143] EXAMPLE 15: Action on the hydration of plant cells

[0144] It is known that the action of salt water on animal or plant tissue leads to dehydration of the cells due to the difference in osmotic pressure between the outside and the inside of the cells of the tissue. The action of two fermented extracts was observed, namely nettle extract E2 and tea extract E5 prepared as described in Example 2.

[0145] A salt water solution (NaCl 0.9%) was added to 1% of nettle extract E2 on the one hand, and 1% of tea extract E5 on the other. A few drops of each preparation were placed on a leaf of porcelain plant (Graptopetalum Paraguayense). The reaction of the plant to the salt water solution is taken as a control. The effects are illustrated in [Fig. 17]. A very clear action on the cell wall is observed: the nettle extract E2 protects against dehydration (photo A), the tea extract E5 promotes hydration (photo B), while the salt water causes a thinning of the walls thus marking its dehydrating effect (photo C). At the cellular level, the two fermented extracts inhibit the dehydrating effect of salt water with a more pronounced action for the fermented tea extract. EXAMPLE 16: Action on germination

[0146] The two extracts of nettle E2 and tea E5 were tested on a tuber of Oca of Peru (Oxalis tuberosa) which has the ability to germinate and grow easily above ground in a humid environment. Three sprouted tubers are placed in tubes containing salt water with or without the addition of fermented extracts E2 or E5. They are incubated in natural light between 20°C and 25°C at approximately 40% humidity with 5 ml of liquid at the bottom of the tube, namely respectively: salt water NaCl 0.9% (tube ES), fermented tea extract E5 1% in salt water NaCl 0.9% (tube Thé-F-1%) and fermented nettle extract E2 1% in salt water NaCl 0.9% (tube Ort-F-1%). Over time, only fresh water is added so that the tuber does not dehydrate. After 30 days, it was observed that the tuber of the Ort-F-1% tube showed growth of around 2.5 cm in length with the appearance of foliage, whereas the leaf stems of the Thé-F-1% and ES tubes had lengthened very little. It was also noted that the tuber of the ES tube showed mold, which was not the case for the tuber of the Ort-F-1% and Thé-F-1% tubes.However, in this last tube, the tuber did not show any foliage growth.

[0147] The contact of the tuber with the nettle extract E2 was therefore very stimulating and protective. The fermented nettle extract according to the invention is of great interest for the protection of plant cells and the induction of their growth. The fermented tea extract, for its part, although it helps to prevent cell dehydration, does not induce growth.

Claims

Claims

1. Process for producing a plant extract, characterized in that it comprises the steps of: a) - preparing a fermentation medium containing a whole plant or a part of a plant constituting a plant substrate in aqueous phase, b) - inoculating said fermentation medium with a preparation of microorganisms formed from a consortium in a sugary aqueous medium, which consortium comprises at least one microorganism belonging to the group of lactic acid bacteria, at least one microorganism belonging to the group of yeasts and at least one microorganism belonging to the group of acetic acid bacteria, c) - incubating in an open reactor, at a temperature between 12°C and 45°C, for 2 to 20 days, d) - recovering the supernatant, with or without the residual biomass, to obtain an aqueous extract rich in active compounds of said plant.

2. Process for producing a plant extract according to the preceding claim, characterized in that said preparation of microorganisms comprises: - at least one lactic acid bacterium belonging to the genus lactobacillus or pediococcus, - at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, - at least one acetic acid bacterium belonging to the genus acetobacter, gluconobacter or komagataeibacter

3. Process for producing a plant extract according to claim 2, characterized in that said preparation of microorganisms comprises: - at least one lactic acid bacterium chosen from Lactobacillus plantarum and Lactobacillus acidophilus, - at least one yeast chosen from Saccharomyces cerevisiae, Saccharomyces boulardii, Schyzosaccharomyces pombe and Torulaspora delbrueckii.

4. A method of producing a plant extract according to claim 2, characterized in that said at least one acetic acid bacterium comes from unpasteurized cider vinegar or wine vinegar.

5. Process for producing a plant extract according to one of the preceding claims, characterized in that the culture medium of said preparation of microorganisms comprises an aqueous extract of a plant or a part of a plant, chosen from an infusion, a decoction, a maceration or a juice.

6. A method of producing a plant extract according to one of the preceding claims, characterized in that the culture medium of said preparation of microorganisms comprises a tea infusion and a source of sugars chosen from glucose, fructose or a mixture thereof.

7. Process for producing a plant extract according to one of the preceding claims, characterized in that said preparation of microorganisms is a cell suspension cultivated in an open reactor, at a temperature between 12°C and 45°C for 6 to 21 days, to obtain a consortium of said microorganisms, comprising a total flora of 105CFU / g to 107CFU / g.

8. Process for producing a plant extract according to one of the preceding claims, characterized in that, in step b), said fermentation medium is inoculated at an inoculation rate of between 102 CFU / g and 104 CFU / g, and incubated at a temperature of between 25°C and 30°C, for 2 to 20 days.

9. Process for producing a plant extract according to one of the preceding claims, characterized in that the plant substrate is chosen from higher plants, lower plants or phytoplankton organisms.

10. Process for producing a plant extract according to one of the preceding claims, characterized in that the plant substrate is chosen from nettle, woad, Damask rose, jujube, Ginkgo biloba, rosemary, lavender, chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina.

11. Process for producing a plant extract according to one of the preceding claims, characterized in that the plant substrate is obtained in the form of fragments, ground material or powder, from a whole plant or part of a plant, or as a solid co-product resulting from a treatment of said plant.

12. Process for producing a plant extract according to the preceding claim, characterized in that in step a), the plant substrate is mixed with water then brought to a temperature between 25°C and 90°C, and if necessary sugar is added, before or after bringing to temperature, to bring the fermentation medium to a Brix degree between 2 and 10.

13. Process for producing a plant extract according to one of claims 1 to 9, characterized in that the plant substrate is obtained from a fresh or dried plant or part of a plant in the form of a juice, an infusion, a decoction or a maceration, or as a liquid co-product resulting from a treatment of said plant.

14. Process for producing a plant extract according to the preceding claim, characterized in that in step a), the plant substrate is brought to a temperature of between 25°C and 85°C, and if necessary, sugar is added before or after bringing to temperature, to bring the fermentation medium to a Brix degree of between 2 and 10.

15. Process for producing a plant extract according to one of the preceding claims, characterized in that in step a), the fermentation medium is sweetened by adding a source of assimilable sugars chosen from sucrose, glucose, fructose, molasses, honey, or a mixture thereof.

16. Process for producing a plant extract according to one of the preceding claims, characterized in that it further comprises, after step d), one or more of the following operations: filtration of the fermented extract to eliminate plant residues, sterilizing filtration to eliminate microbial flora, microbiological decontamination, freeze-drying, drying in an oven, grinding.

17. Plant extract containing active compounds, obtained by a process according to one of the preceding claims.

18. Plant extract according to the preceding claim, characterized in that it is produced from a plant chosen from nettle, woad, Damask rose, jujube, Ginkgo biloba, rosemary, lavender, chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina.

19. Use of a plant extract according to one of claims 17 or 18 or capable of being obtained by a process according to one of claims 1 to 16, for the manufacture of a product

20. pharmaceutical, a cosmetic product, a perfume, a food supplement or a plant treatment product. Use according to the preceding claim for the manufacture of a pharmaceutical product or a cosmetic product additionally comprising at least one compound chosen from: an excipient, a natural additive, a synthetic additive, a thickener, a stabilizer, an emulsifier, an adjuvant, or a mixture thereof.

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