FR3103826A1 - PREPARATION OF MICRO-ORGANISMS FOR THE FERMENTATION OF PLANTS, EXTRACTS OBTAINED AND THEIR USES - Google Patents

Abstract

The invention relates to a preparation of microorganisms comprising, in a sweet 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 bacterium, for example of the genus acetobacter, gluconobacter, or komagataeibacter. It also relates to a process for the production of a plant extract rich in active compounds, by fermentation in the presence of said consortium of a substrate which may be obtained from a whole plant, from a part of a plant, or from 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

PREPARATION OF MICROORGANISMS FOR PLANT FERMENTATION, EXTRACTS OBTAINED AND USES THEREOF

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 the fermentation of plants using this composition and the use of the plant extracts obtained in various applications.

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

Among the conventional 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 the latter. The techniques of extraction by organic solvent, long preponderant, are criticized today because of the pollution induced by these solvents which generate ecotoxic waste whose recycling is difficult and disposal not very respectful of the environment. Moreover, these processes are very energy-intensive and often require specific confinement conditions (supercritical CO ₂ , subcritical water) making their implementation complex. An alternative may be extraction in the oily phase using solvents of natural origin, such as vegetable oils. However, this technique only makes it possible to extract lipophilic compounds. The aqueous phase extraction has low yields, unless special processing conditions are implemented to improve the 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 the operating conditions (in particular pH, ionic strength, pressure, temperature, sterility).

Another approach consists in using plants as raw material for transformations orchestrated by micro-organisms. Many processes involving selected or modified microorganisms are described in the literature and are used in an industrial environment. The most widespread are based on fermentation, known for millennia as a food preservation process and defined as the transformation of carbonaceous substrates in the absence of oxygen. Fermentation is based on the endogenous microflora of the substrate or on added microorganisms, generally chosen for their fermentation capacities or resistance to environmental conditions. In these processes, a type of microorganism transforms a given substrate, for a unique determined objective. For example, we can cite winemaking processes (grapes – yeasts – wine production) or beer brewing (barley malt – yeasts – beer production).

In addition, 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 micro-organisms, 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 costly purification steps, both in terms of energy and purification solvents and waste treatment. In all cases, here again, these processes concern a triptych of substrate – microorganism – single objective.

However, we know that in nature, micro-organisms organize themselves within communities acting in interaction, which allows them to carry out the biodegradation of organic matter, including in unfavorable environments. These communities, called consortia (consortium in the singular), develop synergistic behaviors thanks to close metabolic couplings. Therefore, it appeared interesting to design combinations of micro-organisms chosen in an adequate way 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 ensilage operations. Another promising way is the production of hydrogen by degradation of biomass. Work carried out with a view to optimizing 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 consortium of the floor.

The present invention aims to take advantage of these faculties of cooperation 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 to extract or make available compounds present in a plant substrate, without resorting to synthetic inputs and without rejecting polluting waste. These operating conditions, which are also energy efficient, meet an objective of developing so-called eco-responsible technologies.

An object 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 objective of the invention is to propose a consortium capable of fermenting and transforming all kinds of plant substrates, in order to extract and make accessible the compounds which they contain. Yet another object of the invention is to achieve efficient extraction of the active compounds from various plants, including compounds that are not easily accessible by other techniques, with a high yield. Another objective of the invention is to obtain extracts rich in various active compounds, which can be used as such, or for the subsequent preparation of products for pharmaceutical, cosmetic, food, phytosanitary or other purposes.

The present invention is based on an unprecedented approach, according to which a unique microbial consortium works with a wide variety of plants and with any type of plant substrate or of plant origin, liquid or solid, to extract therefrom numerous compounds available in the form of aqueous or powder extracts, easy to store and use in all kinds of applications.

A first object of the invention thus relates to a preparation of microorganisms comprising a consortium of microorganisms in a sugary 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.

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

Yeasts are aerobic-anaerobic eukaryotic cells that can be described as biological multi-reactors. They use carbonaceous substrates as sources of carbon and energy. They are able to metabolize many sugars, with the exception of polysaccharides. In particular, they produce CO ₂ and alcohol, as well as numerous aromatic molecules.

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 by transforming it into acetic acid. In addition, they are able to synthesize a biofilm, which will play an important role in the stability of the consortium, as we will see later.

Lactic acid bacteria, when they are 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 the acidification of the medium in free forms. It is known that the presence of yeast contributes to their growth.

It is rare that under natural conditions, these three groups find favorable conditions for 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 also 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.

For the lactic acid bacteria, microorganisms of the order Lactobacillales of the Lactobacillaceae family of group 1 are advantageously used, in particular those belonging to the lactobacillus and pediococcus genera. 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.

Yeasts of the order Saccharomycetales of the family Saccharomycetaceae or of the order Schyzosaccharomycetales of the family Schyzosaccharomycetaceae can be advantageously used, in particular those of the genera Saccharomyces, Schyzosaccharomyces or Torulaspora . For example, baker's yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii , beer yeasts Saccharomyces boulardii and Schyzosaccharomyces pombe can be chosen, alone or in combination.

For the acetic acid bacteria, bacteria of the order of the rhodospirillales of the family of the acetobacteraceae are preferably used, with in particular the genera acetobacter, gluconobacter and komagataeibacter ( gluconacetobacter ) . These acetic bacteria may originate from cider vinegar or unpasteurized wine with an acidity level of 4% to 5%, advantageously from commercial cider vinegar, labeled "organic farming" according to the definition of the 'European Union. The inoculation is therefore carried out by direct addition of an aliquot of vinegar to the composition. In vinegars, alongside the dominant acetic bacteria, there is a complex natural indigenous flora, part of which will be able to cohabit with the added micro-organisms. Another part will collapse during the establishment of the consortium, in the same way that the development in symbiosis of all the micro-organisms forming the consortium prevents the development of undesirable species, including pathogenic species.

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 lactobacillus or pediococcus genus,
- at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora,
- at least one acetic bacterium belonging to the genus acetobacter, gluconobacter, or komagataeibacter .

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 bacterium from cider vinegar or wine vinegar.

The culture medium also plays an important role. In the composition according to the invention, the medium is a sugary nutrient medium, that is to say rich in sugars that can be assimilated by the microorganisms present. These carbohydrates, usually mono- or disaccharides, can come from a variety of sources. It can be a pure sugar, usually sucrose, glucose or fructose, or a processed or co-produced product, such as molasses. Mixed sources are possible although more complex to manage. It should be noted that sucrose being assimilable by many microorganisms, and in particular by those of the consortium according to the invention, it will be preferred.

Originally, the aqueous phase of the nutrient medium is not pure water, but a plant extract. It may 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 leaves of tea, coffee beans, cocoa pods, etc. 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 additional 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.

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

This medium, completely vegetable and without synthetic input, ensures the vital comfort of each of the micro-organisms. It offers each of them the possibility of satisfying their 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 sweet nutrient medium, with an inoculation rate commonly comprised between 10 ³ CFU/g and 10 ⁵ 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, after which a composition of microorganisms with a total flora of between 10 ⁵ CFU/g and 10 ⁷ CFU/g. A microbiological count of 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. It is also possible to preserve the composition by refrigerating it at 4° C. or by freezing, with a view to subsequent use.

Thus, according to a preferred embodiment of the preparation of microorganisms which is the subject of the present invention, the latter is obtained by a process comprising:
- introducing said microorganisms into said nutrient medium to obtain a microbial suspension, and
- 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 from 10 ⁵ CFU/g to 10 ⁷ CFU/g.

It has been observed that under the aforementioned 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 special measures to ensure its asepsis vis-à-vis 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 culture under the aforementioned conditions. Indeed, a distribution is created in the reactor which is dependent among other things on the O ₂ gradient of the medium and its redox potential, and also on the appearance of a biofilm produced by the micro-organisms at the water-water interface. air. This biofilm advantageously makes it possible to protect the environment from environmental, chemical or biological contaminants. It materializes the cooperation of the three groups of micro-organisms and contributes to the robustness of the process.

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 they contain.

Thus a second object of the present invention relates to a process for the production of a plant extract, comprising the steps consisting in:
a) - prepare a fermentation medium containing a plant substrate in aqueous phase,
b) - inoculating said fermentation medium with a preparation of microorganisms described above in an amount such that the inoculation rate is between 10 ² CFU/g and 10 ⁴ 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) - recovering the supernatant, with or without the residual biomass, to obtain an extract of said plant rich in active compounds.

The extract obtained can therefore consist of the liquid supernatant alone or of the whole of the culture medium which can be harvested with the biomass and optionally undergo drying.

In a preferred embodiment of the method for producing a plant extract according to the invention, in step b), the fermentation medium is inoculated with an amount 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 between 25° C. and 30° C., for 10 to 15 days. Preferably, the inoculation rate is between 0.8% and 3%.

As can be seen, even inoculated at relatively low rates (ie between 10 ² CFU/g and 10 ⁴ CFU/g), the consortium rapidly 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 various micro-organisms. This allows it to be kept in the culture medium for several days, even a few weeks, without human intervention. Although fermentation takes place in principle in anaerobic conditions, the process is carried out here in an open environment for gas exchanges with the atmosphere, and without any particular constraint 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.

The composition of inoculated microorganisms is capable of directing the extractive fermentation of all kinds of plants. The substrate can be chosen from higher plants of any type, whether flowering plants, aromatic plants, cereals, fodder plants, protein crops, oilseeds, or others, representing a great diversity of compounds likely to be used and recovered. It can also belong to the lower plants, among which we find ferns, mosses, fungi, lichens and algae. It can also be chosen from the organisms found in phytoplankton, that is to say all the 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.

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 unicellular organisms such as spirulina. Whatever the substrate, we will use as much as possible sources of supply respecting the standards of organic farming which refuses the use of synthetic chemicals.

According to particular embodiments of the invention, the plant substrate may be nettle ( Urtica dioica ), pastel ( 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 chamomile, chamomile…), heather (especially the species Calluna vulgaris ), ginger ( Zingiber officinale ), turmeric ( Curcuma longa ), coffee ( Rubiaceae coffea ), tea ( Camellia sinensis ), cocoa ( Theobroma cacao ), or even spirulina ( Spirulina ).

The plant substrate can include a whole plant or a part of a plant. It can also be fresh or dried, native or processed. The part of the plant chosen can be that which is known in the state of the art as containing compounds of interest or possessing particular properties, such as turmeric root, Jujube fruit, lavender flower, etc. . We can also choose other parts of the plant, which will be able to reveal their content of active compounds neglected, even ignored until today, but which it is possible to extract and valorize thanks to the synergistic action of the consortium. microbial according to the invention. Plant by-products can also be used: the leaves of pastel 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 may be in a solid form or in a form having a preponderant liquid component. Their implementation will be adapted accordingly.

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 from a plant part, or a solid co-product resulting from a treatment of said plant for other purposes and in other processes.

In accordance with 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 it to temperature, to bring the fermentation medium to a Brix degree of between 2 and 10. The sugar supply can be up to 100 g/l of fermentation medium, or be useless when the substrate itself is sufficiently sweet.

According to a second embodiment of the invention, the plant substrate can 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 fresh or dried plant or part of a 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 other equally interesting ones.

According to a mode of production of 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 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. The sugar supply can go up to 100 g/l of fermentation medium if necessary . For example, the liquid substrate added with 50 g/l of sucrose is heated to a temperature of the order of 70° C., for 5 min to 10 min. A fraction of dechlorinated water can be added to dilute the juices in certain cases that those skilled in the art will be able to identify.

In the cases where in step a), the addition of sugar is required to bring the fermentation medium to the desired rate, 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. Note that 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.

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

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

These filtrations will be omitted if the biomass is kept, as is the case when seeking to recover active ingredients that have remained in the biomass, but have been made bio-available by fermentation and the action of the enzymatic equipment of the consortium's micro-organisms . It is specified that in this description, the expression "plant extract" refers to the product resulting from the process of extraction by fermentation, with or without filtration, and both with and without the plant and microbial biomass of the fermentation medium.

Whether the plant extract is a solute or a mass, we will make sure to stop the 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.). It is possible to freeze-dry the aqueous extracts, dry the solid extracts, for example by passing them through an oven between 30° C. and 45° C., and grind them more or less finely depending on the intended use.

The process according to the invention may 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 the microbial flora, microbiological decontamination, freeze-drying, drying in an oven, grinding. The person skilled in the art will know how to choose the appropriate techniques depending on the initial state of the extract and the desired form with a view to its conservation and subsequent use.

The method which has just been described constitutes a significant saving in time and reduces the operations involving operators. It meets current requirements in terms of eco-responsibility: no synthetic input, culture conditions in an open system low in energy and without asepsis, operating autonomy during the process. It is industrializable and economically profitable. It is emphasized that it allows access to compounds that are rarely extracted by classical 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 supramolecular structures of the plant, of simplifying macro-compounds to make them soluble and thus release them. Therefore, the process can be described as an extraction-fermentation-transformation process (all in one).

A plant extract containing active compounds, obtained by a method as described above is also a subject of the present invention. As explained above, this extract can be produced from a multitude of plants, the plant possibly being chosen from higher plants, lower plants or phytoplankton organisms. May be concerned 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, pastel, rose damask, jujube, ginkgo biloba, rosemary, lavender, chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina.

As explained above, the plant extracts obtained using the composition of microorganisms by the extraction process according to the invention are characterized by a great diversity of compounds obtained, and as a corollary a great variety of activities biological properties, physico-chemical properties and organoleptic properties, in particular olfactory, with in certain cases, interesting synergies. They are therefore particularly suitable for use in compositions developed for the implementation of 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 a fermented plant extract 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, an additive synthetic, a thickener, a stabilizer, an emulsifier, an adjuvant, or a mixture thereof.

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

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 skin application, in particular a cream. This cream can for example comprise one or more fermented plant extracts such as a nettle extract, an extract of ginkgo biloba, an extract of pastel, or others.

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 fragrant extract that the expert will imagine. to assemble.

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.

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 stimulation of plant growth.

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

represents the evolution of the iron content in a nettle extract according to the invention.

represents the evolution of the total acidity in the same nettle extract.

presents the rate of living fibroblasts at different concentrations of nettle extract E1.

presents the rate of living keratinocytes at different concentrations of the E1 extract.

presents the influence of this nettle extract on different activities.

shows the level of living fibroblasts at different concentrations of the Ginkgo biloba E3 extract according to the invention.

presents the rate of living keratinocytes at different concentrations of the same Ginkgo biloba extract.

presents the influence of this E3 extract on different activities.

gives the absorption spectra of Turmeric extracts according to the invention.

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

presents the rate of living keratinocytes at different concentrations of the same extract.

shows the rate of living fibroblasts at different concentrations of the E7 extract (juice co-produced from pastel) according to the invention.

presents the rate of living keratinocytes at different concentrations of the same E7 extract.

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

presents the rate of living keratinocytes at different concentrations of the same E8 extract.

shows the influence of the E6 extract on different activities.

presents the influence of the E7 extract on different activities.

presents the influence of the E8 extract on different activities.

gives the absorption spectra of a spirulina fermentation medium according to the invention.

represents the effect of extracts according to the invention on plant cells.
EXAMPLE 1: Elaboration of a composition of microorganisms

The selected micro-organisms 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. Sucrose can be replaced by a mixture of glucose and fructose. The inoculation rate is between 10 ³ CFU/g and 10 ⁶ CFU/g of medium depending on the groups of microorganisms. The acetic acid bacteria are brought by an amount of unpasteurized vinegar such that the pH of the nutrient medium remains in an interval of 4 to 5. The microorganisms grow easily under the 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 of cylindrical shape, so that the system is dynamic and self-regulation takes place. After a fortnight, a biofilm forms indicating the creation of the consortium. The consortium is maintained in the nutrient medium while respecting a consortium/medium mass ratio ranging from 50/50 to 20/80. After about 3 weeks, the pH and the Brix degree have decreased indicating that the sugars are consumed. Subculturing on fresh nutrient medium is then carried out according to the conventional techniques known in the state of the art.

Compositions of microorganisms were developed according to this procedure.

Composition C1 : Composition C1 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 5% unpasteurized wine vinegar of acidity, in an infusion of black tea prepared with 8 g/l of leaves infused for 15 min at 85°C then filtered, and added with 50 g/l of sucrose. The biofilm of the consortium is formed from 15 days.

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 5% unpasteurized cider vinegar of acidity. The ferments are introduced into an infusion prepared with 10 g/l of tea leaves infused for 20 min at 80° C. then filtered, and added with 70 g/l of sucrose. The biofilm of the consortium is formed from 15 days, although a little less important than in composition C1.

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 between 10 ⁶ CFU/g and 10 ⁷ CFU/g, with a similar representation of each microbial group. For comparison, each microorganism of composition C1 was cultured individually under the same conditions: sweet tea culture medium, an inoculation rate close to 10 ⁶ CFU/g, reactors under non-aseptic conditions , temperature from 25°C to 30°C. After 5 days, the lactobacilli did not survive and the yeasts were contaminated. The test with vinegar alone did not show any change: neither contamination nor growth of acetic bacteria. Furthermore, sweetened black tea identical to the nutrient medium of composition C1 was incubated under the same conditions. It showed mold contamination after 5 days. None of the trials showed biofilm formation.

EXAMPLE 2: Preparation of plant extracts

Extract E1
A powder of nettle ( Urtica dioica ) is mixed with dechlorinated water heated to 80° C. in the amount of 50 g/kg of medium. Sucrose is then added up to 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. The nettle extract E1 is obtained, with the nettle powder residue. The solid and liquid fractions can then be separated by filtration, centrifugation or another technique known per se, depending on the applications envisaged.

Extract E2
A nettle powder (Urtica dioica) is mixed with dechlorinated water heated to 80°C at 50 g/kg of medium. The medium is then sweetened to the tune of 50 g/kg of medium and left to cool. Composition C2 prepared according to Example 1 is added in the proportions of 2% to the aqueous suspension of unfiltered nettle. 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 eliminate plant residues and a second sterilizing filtration on 0.2 µm to eliminate ferments. The extract E2 is obtained.

Extracts of rosemary ( Rosmarinus officinalis ), damask rose ( Rosa damascena ) and jujube leaves ( Ziziphus zizyphus ) were prepared according to an identical protocol.

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

Extract E4
Turmeric ( Curcuma longa , root powder) is put 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 collected and dried. A sample is taken and taken up in water at 200 g/kg to characterize it. It is compared to unfermented turmeric powder and taken up in water at 200 g/kg.

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

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 up to 50 g/l. After cooling to room temperature, composition C1 prepared in Example 1 is added at 2%. The whole is incubated at 25° C. for 14 days. The liquid phase acidified to 2% (total acidity in acetic acid equivalent) is sampled and then filtered through a 0.2 μm filter.

Extract E6
The extraction of the pastel blue pigment taken 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 with 1 ton of fresh leaves, 1 kg of pigment is extracted. The pastel-targeted extraction process therefore generates co-products in large quantities, 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, the fermentation of Isatis leaves without control gives rise to the development of putrid flora giving off pestilential odors during the spreading or storage of this waste. We were interested in three co-products, namely the fresh leaves after decoction in water (E6 extract), the liquid phase resulting from the extraction of the pastel obtained after precipitation and decantation of the pastel pigment (E7 extract) and the juice from the decoction of fresh leaves (E8 extract).

Isatis leaves having undergone a first extraction by decoction were used to prepare the fermented extract E6. To do this, 8.5 kg of wet whole leaves (containing about 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 the latter reaches 30° C., composition C2 prepared according to Example 1 is added at 2%. The whole is incubated for 10 days at 28°C. The fermentation supernatant is removed and then filtered to remove plant residues, then sterile over 0.2 µm to remove ferments.

The extract obtained has a fresh and vegetable smell, characteristic of Isatis, whereas the initial microbial load of the wet leaves was high since it was greater than 3.10 ⁷ CFU/g. The fermentation controlled and orchestrated by the consortium of microorganisms of the C2 composition therefore made it possible to overcome the endogenous flora of the leaves of Isatis. The minerals released during the fermentation were determined by inductively coupled plasma spectrometry (ICP-OES) or by inductively coupled mass spectrometry (ICP-MS). The rate of calcium released is 218.0 mg/kg, that of magnesium 31.4 mg/kg and that of iron 9.15 mg/kg. These mineral concentrations are significant and demonstrate the interest of fermenting the wet leaves resulting from the decoction for their decomplexation and their release.

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 thrown away, by treating it by fermentation. The juice was heated to 80°C and, at this temperature, sucrose is added at 50 g/l and maintained at this temperature for 15 min in order to reduce the endogenous microbial load which amounts to 5.10 ⁷ CFU/ g. When the medium is cooled, composition C2 is added at 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 conducive 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 vegetable odor.

Extract E8
The juice obtained by decoction of the native leaves of Isatis 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 min at 80°C, then sucrose is added at 50 g/l. The sugar juice is maintained at this temperature for 15 minutes. After cooling, composition C2 is added at 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.

The extract obtained is brown-green in color with a characteristic vegetable smell, and foaming. The minerals released during the fermentation were determined by inductively coupled plasma spectrometry (ICP-OES) or by inductively coupled mass spectrometry (ICP-MS). The rate of calcium released 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 exploited for mild cleansing products. It is also noted that the E6 extract obtained from leaves resulting from the production of pastel 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 valorization of the co-products of the pastel is to be considered.

Excerpt 9
Spirulina is a cyanobacterium used for human health because of its richness in micronutrients (minerals, vitamins, amino acids, pigments, essential fatty acids). It is also known for its phycocyanin content, a blue pigment with many biological properties (antioxidant, anti-inflammatory, hepatoprotective, etc.). Its extraction currently requires complex processes involving multiple steps, solvents or synthesis adjuvants or energy-intensive processes (ultrasound, microwave, vortex, freezing/thawing). Spirulina extraction-fermentation was performed to release the phycocyanin and increase its shelf life in wet form.

Spirulina in the form of a dry powder at 20 g/l is put in dechlorinated water heated to 80°C. Sucrose is added at 50 g/kg. Composition C1 prepared according to Example 1 is added to the aqueous suspension of spirulina at 2%. The test is incubated for 6 days at 28°C.

Extract E10
Calluna or common heather is known for its calming, antiseptic and antioxidant properties linked to the presence of tannins and polyphenols such as quercetin. Commercial preparations are alcoholic extracts likely to cause side effects on health and during preparation, on the environment. Extraction by fermentation in a totally aqueous medium offers a solution to this problem. It is also proposed 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.

The fermentation of heather flowers (calluna vulgaris ) was carried out in the presence of chestnut honey. The flower buds 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 below 45°C, chestnut honey is added up to 70 g/l. When the temperature is below 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 acid level 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 can be seen that the consortium according to the invention performed the extractive fermentation perfectly, despite the difficult conditions.
EXAMPLE 3: Extraction of iron from a nettle extract

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

Samples of the supernatant of the three media are taken during the fermentation on D0, D3, D6 and D12 and the iron is assayed by colorimetry with potassium permanganate. The results obtained are presented in FIG. 1. The samples with ORT-AR residues show a level of extracted iron that increases over time, whereas this level does not change in the medium without ORT-SR nettle powder. This attests to the continuous extraction of iron from plant residues during fermentation, the maximum extraction rate being reached from the ^6th day by the action of added ferments. Indeed, iron extraction is also visible in the samples without ORT-SSF ferment, but it is slower since it only reaches its maximum after 12 days. On the other hand, the ORT-SSF samples show a significant proliferation of the native flora of the nettle, resulting in 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 vegetable smell. The 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 Figure 2. The sugar level in degrees Brix was recorded on D0 and D6.

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 the production of acid has stopped, thus indicating the absence of fermentation strictly speaking, whereas the sugars are not completely consumed (on D6, the sugar is dosed 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 at D6, against 5.3 at D0; for the ORT-SR samples, the sugar is at 2.7°Bx at D6, against 5.1 at D0). The production of acid with consumption of sugar during the fermentations is characteristic of the establishment of the consortium of the C2 composition in the fermentation media.

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 the wild microbial development, protecting both the liquid extract and the residual biomass of the plant as well treated.
EXAMPLE 4: Biological properties of a nettle extract

Cytotoxicity
The E2 nettle extract obtained in Example 2 was tested with regard to 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 suitable for each type of cell in concentrations of between 0.01% and 10%. The diluted extracts are brought into 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 coloration) is proportional to the number of living cells. The results given in Figure 3 (fibroblasts) and Figure 4 (keratinocytes), show that the E2 extract does not show any cytotoxicity compared to the control without extract, on the two cell types, when it is present at a content between 0.01% and 1% in vitro , which is compatible with cutaneous applications.

Cell activities
The E2 extract was then tested on a genome chip to identify the stimulated activities. The extract is diluted beforehand in culture medium, at a non-toxic concentration of 0.01%, then incubated with normal human epidermal keratinocytes for 24 hours. The messenger RNAs (mRNA) are extracted then analyzed by RT-qPCR (Real Time quantitative Polymerase Chain Reaction). The results are presented in Figure 5. They are expressed in %, positive in the event of overexpression and negative in the event of inhibition with respect to the expression of the gene in cells cultured under normal conditions.

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 of interest for cosmetic applications of the "anti-aging" type.
EXAMPLE 5: Biological properties of an extract of Ginkgo biloba

Cytotoxicity
The Ginkgo biloba E3 extract obtained in Example 2 was tested with regard to 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 Figure 6 (fibroblasts) and Figure 7 (keratinocytes), show that the E3 extract does not show 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.

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

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 is observed. On the other hand, the activity of the metalloprotease is inhibited. The expression modifications of these genes are of interest for cosmetic applications aimed at reinforcing the barrier function of the skin.
EXAMPLE 6: Extraction of curcumin and derivatives from an extract of Curcuma longa

We know that turmeric is naturally rich in curcuminoids, mainly curcumin. The E4 extract prepared in Example 2, the visual observation of which 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 dried in an oven 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 unfermented native turmeric taken up in water (turmeric-H ₂ O). To this end, the turmeric powders, fermented and native, are taken at 100 mg/ml and ¼ dilutions are made to be able to compare the samples. The samples are centrifuged and the supernatant is analyzed by visible spectrophotometry. The spectra obtained are shown in Figure 9.

The spectra of the three samples show the presence of curcumin (absorbing between 400 nm and 430 nm) and other compounds resulting from the process (absorbing between 450 nm and 500 nm). Curcumin content appears to be equivalent in fermented turmeric supernatant (Curcuma-F-jus) and native turmeric (Curcuma-H2O) showing that fermentation did not degrade this compound. In addition, fermented turmeric powder (Curcuma-F-powder) has very marked curcumin peaks and metabolizing compounds, about four times greater than those of native turmeric. The extraction-fermentation process has 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.
EXAMPLE 7 Biological Properties of Pastel Extracts and Pastel Co-Products

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.

Cytotoxicity
The E6, E7 and E8 extracts were tested with regard to their in vitro cytotoxicity on human keratinocytes and fibroblasts, according to the procedure described in Example 4, with concentrations of extracts comprised between 0.01% and 5%. The results relating to the E6 extract (fermented Isatis leaves, noted FISAF) are given in FIG. 10 (fibroblasts) and in FIG. 11 (keratinocytes). The results relating to the E7 extract (fermented Isatis juice, denoted JISAF) are given in FIG. 12 (fibroblasts) and in FIG. 13 (keratinocytes). The results relating to the E8 extract (untreated leaves, denoted DECISA) are given in FIG. 14 (fibroblasts) and in FIG. 15 (keratinocytes). They show that the E6 extract shows no cytotoxicity compared to the control when it is present at a content of 0.01% to 2.5% in vitro , the E7 and E8 extracts being devoid of toxicity at a content of from 0.01% to 0.5%. Cutaneous applications can therefore be envisaged.

Cell activities
The E6, E7 and E8 extracts were then tested on a genome chip to identify the stimulated activities. The extracts were diluted in culture medium suitable for keratinocytes, at a non-cytotoxic concentration of the extract, then incubated with normal human epidermal keratinocytes for 24 hours. The messenger RNAs (mRNA) are extracted and then analyzed as in example 4. The results are presented in Figures 16, 17 and 18 respectively for the extracts E6, E7 and E8.

The E6 extract shows a marked overexpression of the genes corresponding to aquaporin 3 and to involucrin, and a particularly high overexpression of the genes of the peptidase 3 inhibitor (approximately 350%). Concerning the E7 extract, 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 the E8 extract, several genes are overexpressed between 80% and 180%. The overexpression of the decorin and involucrin genes around 250% is particularly high.

It can be affirmed that the activities detected on the three extracts based on Isatis are of interest for cosmetic applications. It is further observed that the activities detected are different for each of the extracts.
EXAMPLE 8: Extraction of pigments from a spirulina extract

The E9 extract prepared in Example 2 was analyzed. On days D0, D3 and D6, an aliquot of spirulina is removed and centrifuged for spectrophotometric analysis. The spectrum obtained is presented in Figure 16. The spectrum at 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. nm for phycocyanin. Biofilm also contains phycocyanin.

On the other hand, it is known that spirulina is sensitive to microbial contamination in aqueous medium due to its rich composition of nutrients 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 presents a wild contamination, identified by the nauseating odor of putrefaction. On the contrary, in the test carried out with composition C1, the unpleasant marine odor disappeared. It is therefore found that the process according to the invention has made it possible to avoid wild fermentation of the medium.

Thus, the extraction/fermentation makes it possible to extract compounds of interest from the spirulina and also makes it possible to sanitize the spirulina in the aqueous phase. Therefore, it is possible to preserve it without adding preservatives. It can be used as a source of assets, or else. These operations can be carried out without inconvenience in the presence of the biomass of the consortium of microorganisms.
EXAMPLE 9 Care cream comprising a nettle extract

Nettle is generally used for its soothing properties on the skin. The activities present in the E2 extract demonstrated in Example 3 are advantageous for use in a care cream with a restructuring, firming and moisturizing effect. A care cream formulation with fermented nettle extract has been produced and has shown that it is possible to incorporate it easily.

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 copolymer of hydroxyethyl acrylate and sodium acryloyldimethyl taurate) is added with stirring. From 1 g to 5 g of fermented nettle E2 extract (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). Under strong agitation with the deflocculating blade. The oily phase is slowly added 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 emulsion thus obtained is adjusted to a pH between 5 and 5.5 with 1N soda and to a mass of 50 g with demineralized water.

The cream obtained is smooth, slightly shiny and moderately compact. The composition comprising 10% E2 extract is very slightly off-white with a slight characteristic odor of nettle. It spreads easily on the skin, penetrates easily and leaves a silky and comfortable touch.
EXAMPLE 10 Care cream and gel comprising an extract of Ginkgo biloba

Ginkgo biloba is generally used for its effects on blood microcirculation. The activities present in the E3 extract demonstrated in Example 4 are advantageous for use on the skin. The E3 extract was easily incorporated into a care cream and into a gel.

Care cream with Ginkgo biloba extract
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 copolymer of hydroxyethyl acrylate and sodium acryloyldimethyl taurate) is added with stirring. From 0.5 g to 5 g of fermented Ginkgo biloba E3 extract (1% to 10% of the final formula) are added to the previous mixture, which constitutes the aqueous phase. The oily phase prepared as in the previous example is added slowly to the aqueous phase, together with a preservation system. The emulsion thus obtained is adjusted to a pH of between 5 and 5.5 with 1N sodium hydroxide and to a mass of 50 g with demineralised water.

The cream obtained, comprising 10% of E3 extract, is quite compact, shiny white. It does not exhale the typical smell of the plant. It spreads easily and penetrates leaving a silky touch and a well hydrated and clearer skin.

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 has completely dissolved and thickened. From 0.5 g to 5 g of fermented Ginkgo biloba E3 extract (1% to 10% of the final formula) is added with medium stirring. A preservative, for example dehydroacetic acid and benzyl alcohol, is added. The mixture obtained is adjusted to a pH of 5 to 5.5 if necessary, and in mass to 50 g with demineralised water.

The gel obtained has a very gelled texture which spreads well on the skin.

EXAMPLE 11: Anti-itch lotion

The E10 extract based on heather and honey has been tested for its effects on insect bites and in particular tiger mosquitoes. A few drops of pure extract were applied by light massage to the skin of patients presenting with pimples in several places on the body, signs of an inflammatory reaction following mosquito bites. Patients said they no longer felt itching only 3 min to 5 min after application. After 24 hours, the pimples had all clearly regressed, or even completely disappeared. This result shows that the E10 extract has marked antiallergic and soothing properties, which can be used in an aqueous lotion.

EXAMPLE 12: Creation of perfumes

Perfumes in general are made up of an alcoholic part and an aqueous part in varying 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. Water and alcohol (ethanol) being 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 skincare formulations. The mixture of scents and the creation will be the work of the expert.

Different compositions have been 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 the E2 extract of Example 2. The E5 black tea extract prepared according to Example 2 is also employed.

Perfume composition A
A perfuming composition A was produced by mixing fermented extracts of rosemary (5%), Damascus rose (54%), nettle (27%) and jujube leaves (14%). The dominant top note is that evoking the rose supported by the rosemary that we perceive in a second time. To soften the whole, more subtle notes are brought by the other extracts.

Perfuming compositions B1 and B2
Two compositions were made from a commercial eau de toilette with a very sweet and heavy peach scent. In composition B1, was added rosemary extract (very fragrant) up to 20%. In composition B2, 20% fermented tea (low odor 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 B1 and B2 flavors obtained are enlivened by the presence of fermented extracts and the acidity brings volatility to the aromas, thus making them lighter and fresher.
EXAMPLE 13: Treatment of lesions on plants

The effects of a fermented tea extract are shown in an experiment on grape leaves. The vine leaf is damaged in two places with a scalpel (1 and 2). On lesion 1, a drop of the E5 black tea extract described in Example 2 was deposited, lesion 2 is left as it is. A control is constituted by depositing a drop of the E5 extract on a part of the healthy leaf without lesion. After 2 days, there is a very clear change in the plant tissue of the injured area treated with the E5 extract on which the area of the lesion is delimited by diffusion of the E5 product and where the various 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 the stomata of plants

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

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 paraguayense ). The cells observed under an optical microscope (magnification x 600) show widely open stomata. Thus, the application of the fermented tea extract induces a mechanical response of plant cells and facilitates cellular exchanges with their environment, as would blue light on a plant, thus accelerating cell growth. This E5 extract promotes tissue receptivity while maintaining healthy ground.
EXAMPLE 15: Action on the hydration of plant cells

It is known that the action of salt water on an animal or plant tissue causes 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 the nettle extract E2 and the tea extract E5 prepared as described in example 2.

A salt water solution (NaCl 0.9%) was added on the one hand with 1% of nettle extract E2, and on the other hand with 1% of tea extract E5. A few drops of each of the preparations were placed on a porcelain plant leaf ( Graptopetalum paraguayense ). The reaction of the plant to the salt water solution is taken as a control. The effects are illustrated in Figure 17. A very clear action is observed on the cell wall: nettle extract E2 protects against dehydration (photo A), tea extract E5 promotes hydration (photo B) , while salt water leads to a thinning of the walls, thus marking its dehydrating effect (photograph 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

The two extracts of nettle E2 and tea E5 were tested on an Oca tuber from Peru ( Oxalis tuberosa ) which has the ability to germinate and grow easily above ground in a humid environment. Three germinated tubers are placed in tubes containing salt water with or without added 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% (ES tube), extract of 1% E5 fermented tea in 0.9% NaCl salt water (Tea-F-1% tube) and 1% E2 fermented nettle extract in 0.9% NaCl salt water (Ort-F-1 tube %). Over time, only fresh water is added so that the tuber does not become dehydrated. It is observed after 30 days that the tuber of the Ort-F-1% tube shows a growth of the order of 2.5 cm in length with the appearance of foliage, whereas the leaf stems of the Thé-F-1% tubes and ES elongated very little. It is also noted that the tuber of the ES tube shows mould, which is not the case for that of the Ort-F-1% and Thé-F-1% tubes. However, in this last tube, the tuber showed no leaf growth.

The contact of the tuber with the E2 nettle extract 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. Fermented tea extract, on the other hand, while it helps prevent cell dehydration, does not induce growth.

Claims (20)

Preparation of microorganisms characterized in that it comprises a consortium of microorganisms in a sugary aqueous nutrient medium, said consortium comprising 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. Preparation of microorganisms according to claim 1, characterized in that it comprises:
- at least one lactic acid bacterium belonging to the lactobacillus or pediococcus genus,
- at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora,
- at least one acetic bacterium belonging to the genus acetobacter, gluconobacter or komagataeibacter . Preparation of microorganisms according to the preceding claim, characterized in that it 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 bacterium from cider vinegar or wine vinegar. Preparation according to one of the preceding claims, characterized in that the nutrient medium comprises an aqueous extract chosen from an infusion, a decoction, a maceration or a juice of a plant or of a part of a plant. Preparation according to one of the preceding claims, characterized in that the nutrient medium comprises a tea infusion and a source of assimilable sugars chosen from sucrose, glucose, fructose, molasses, or a mixture thereof. Preparation according to one of the preceding claims, obtained by a process comprising:
- introducing said microorganisms into said nutrient medium to obtain a microbial suspension, and
- 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 consortium of said microorganisms, comprising a total flora of 10 ⁵ CFU/g to 10 ⁷ CFU/g. Process for the production of a plant extract, characterized in that it comprises the steps consisting in:
a) - prepare a fermentation medium containing a plant substrate in aqueous phase,
b) - inoculating said fermentation medium with a preparation of microorganisms according to one of the preceding claims at an inoculation rate of between 10 ² CFU/g and 10 ⁴ 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) - recovering the supernatant, with or without the residual biomass, to obtain an extract of said plant rich in active compounds. Process for the production of a plant extract according to the preceding claim, characterized in that in step b), the said fermentation medium is inoculated with a quantity of the said preparation of microorganisms comprised between 0.5% and 5% by mass relative to the mass of fermentation medium, and incubated at a temperature between 25°C and 30°C, for 10 to 15 days. Process for the production of a plant extract according to claim 7 or 8, characterized in that the plant substrate is chosen from higher plants, lower plants or phytoplankton organisms. Process for the production of a plant extract according to one of Claims 7 to 9, characterized in that the plant substrate is chosen from nettle, pastel, Damask rose, jujube, Ginkgo biloba , rosemary, lavender, chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina. Process for the production of a plant extract according to one of Claims 7 to 10, characterized in that the plant substrate is obtained in the form of fragments, ground material or powder, from a whole plant or from a part of a plant, or as a solid co-product resulting from a processing of said plant. Process for producing a plant extract according to the preceding claim, characterized in that in step a), the plant 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 heating, to bring the fermentation medium to a Brix level between 2 and 10. Process for the production of a plant extract according to one of Claims 7 to 10, characterized in that the plant substrate is obtained in the form of a juice, an infusion, a decoction or a maceration, from a fresh or dried plant or part of a plant, or as a liquid co-product resulting from a treatment of said plant. Process for the production of 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 an addition of sugar is carried out before or after bringing to temperature, to bring the fermentation medium to a Brix degree between 2 and 10. Process for the production of a plant extract according to one of Claims 7 to 14, characterized in that in stage a), the fermentation medium is sweetened by adding a source of assimilable sugars chosen from sucrose, glucose , fructose, molasses, honey, or a mixture thereof. Process for the production of a plant extract according to one of Claims 7 to 15, characterized in that it further comprises, after stage 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. Plant extract containing active compounds, obtained by a process according to one of Claims 7 to 16. Plant extract according to the preceding claim, characterized in that it is produced from a plant chosen from higher plants, lower plants or phytoplankton organisms. 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 7 to 16, for the manufacture of a pharmaceutical product, a cosmetic product, a perfume, food supplement or plant treatment product. Use according to the preceding claim for the manufacture of a pharmaceutical product or a cosmetic product comprising at least in addition a compound chosen from: an excipient, a natural additive, a synthetic additive, a thickener, a stabilizer, an emulsifier, an adjuvant , or a mixture thereof.