WO2021111286A1

WO2021111286A1 - Preparation of micro-organisms for extracting active plant ingredients, extracts obtained and their uses

Info

Publication number: WO2021111286A1
Application number: PCT/IB2020/061310
Authority: WO
Inventors: Frédérique CAUZAC; Valérie REYNAUD
Original assignee: Biocorium
Priority date: 2019-12-02
Filing date: 2020-12-01
Publication date: 2021-06-10
Also published as: FR3103826A1

Abstract

Method for producing an aqueous plant extract rich in active compounds, comprising the steps of: a) - preparing a fermentation medium which contains a plant substrate in the aqueous phase, b) - inoculating the medium with a preparation of micro-organisms which form a grouping which comprises at least one micro-organism which belongs to the group of lactic acid bacteria, for example Lactobacillus or Pediococcus, at least one micro-organism which belongs to the group of yeasts, for example Saccharomyces, Schizosaccharomyces or Torulaspora, and at least one micro-organism which belongs to the group of acetic acid bacteria, for example Acetobacter, Gluconobacter, or Komagataeibacter, c) - incubating in an open reactor, d) - retrieving the supernatant, with or without the residual biomass. Using the extracts obtained to manufacture pharmaceutical products, cosmetics, perfumes, food supplements or products for treating plants is also claimed.

Description

PREPARATION OF MICRO-ORGANISMS FOR THE EXTRACTION OF PLANT ACTIVE INGREDIENTS, EXTRACTS OBTAINED AND THEIR USES

The present invention belongs to the field of the extraction of active compounds of plant origin and their valuation. It relates to a process for the fermentation of plants using a composition of microorganisms 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 exhibiting particular biological activities. The use of these substances supposes to extract them from the plant tissues containing them and to make 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 methods in which the plant material rich in the compound of interest is immersed in a liquid having an affinity therewith. Organic solvent extraction techniques, which were predominant for a long time, are now criticized because of the pollution caused by these solvents which generate ecotoxic waste, the recycling of which is difficult and the elimination not respectful of the environment. In addition, these processes are very energy intensive and often require special confinement conditions ( _{supercritical CO 2} , subcritical water) making their implementation complex. An alternative may lie in the oil phase extraction using solvents of natural origin, such as vegetable oils. However, this technique only makes it possible to extract lipophilic compounds. The extraction in the aqueous phase has low yields, unless special treatment 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 in order to ensure sufficient storage by strict control of the operating conditions (in particular pH, ionic strength, pressure, temperature, sterility).

Another approach is to use plants as the raw material for transformations orchestrated by microorganisms. Numerous 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 method of preserving food and defined as the transformation of carbon 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 single determined objective. For example, we can cite the processes of winemaking (grapes - yeasts - wine production) or beer brewing (barley malt - yeasts - beer production).

In addition, biotechnological processes involving 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 contaminants). They are followed by expensive purification steps, both in energy, purification solvents and waste treatment. In all cases, here again, these methods relate to a triptych substrate - microorganism - single objective. However, we know that in nature, microorganisms organize themselves in communities acting in interaction, which allows them to achieve the biodegradation of organic matter, including in less favorable environments. These communities, called consortia (consortium in the singular), develop synergistic behaviors thanks to close metabolic couplings. Consequently, it appeared interesting to design combinations of microorganisms chosen in an adequate manner 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 route is the production of hydrogen by degradation of biomass.

Work carried out with a view to optimizing the production of bio-hydrogen has demonstrated the existence of a physical interaction and exchanges of cytoplasmic molecules between the microbial cells of a consortium selected from a natural soil consortium.

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 aim of the present invention is thus to provide a consortium of microorganisms, defined and stable, capable of reacting with a plant substrate in an aqueous medium in a process making it possible to ferment and transform all kinds of plant substrates, in order to extract and render accessible the compounds they contain. Another objective of the invention is to achieve an efficient extraction of the active compounds from different plants, including compounds which are not easily accessible by other techniques, with a high yield. Another objective of the invention is to carry out an extraction of compounds hitherto not isolated or even not identified. 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, plant protection or other use.

The present invention is based on a novel 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.

DISCLOSURE OF THE INVENTION

A first object of the invention thus relates to a process for producing a plant extract, comprising the steps consisting in: a) - preparing a fermentation medium containing a plant substrate in aqueous phase, b) - inoculating said fermentation medium with a preparation of microorganisms forming a consortium in a sweet 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 bacteria, 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 aqueous extract rich in active compounds of said plant.

The microorganisms making up the artificial consortium 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 facultative aerobic-anaerobic eukaryotic cells that can be described as biological multi-reactors. They use carbon substrates as sources of carbon and energy. They are able to metabolize many sugars, with the exception of polysaccharides. In particular, they produce CO2 and alcohol, as well as many aromatic molecules.

Acetic bacteria consume simple sugars to produce organic acids such as gluconic acid and vitamins, among other things. 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 discussed below.

Lactic 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 environment in free forms (planktonic). We know that the presence of yeasts contributes to their growth.

Rarely, 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. This assembly is also designed in such a way as to combine complementary activities. In the composition present, the genera of microorganisms have been favored, within which particular species have been retained as more favorable to these multiple requirements.

For lactic acid bacteria, use is advantageously made of microorganisms of the order Lactobacillales of the Lactobacillaceae family of group 1, 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.

Yeasts of the order Saccharomycetales of the Saccharomycetaceae family or of the order Schizosaccharomycetales of the Schizosaccharomycetaceae family can be advantageously used, in particular those of the genera Saccharomyces, Schizosaccharomyces or Torulaspora. For example, baker's yeast Saccharomyces cerevisiae, and Torulaspora delbrueckii, brewer's yeast Saccharomyces boulardii and Schizosaccharomyces pombe can be chosen, alone or in combination.

For acetic 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 bacteria can originate from an unpasteurized cider vinegar or wine having an acidity level of 4% to 5%, advantageously from a commercial cider vinegar, labeled "Organic farming" according to the definition of l 'European Union. The inoculation is therefore carried out by direct addition of an aliquot of vinegar in the composition. In vinegars, alongside the dominant acetic bacteria, there is a complex natural native flora, part of which will be able to coexist with the added microorganisms. Another part will collapse during the establishment of the consortium, in the same way that the development in symbiosis of all the microorganisms forming the consortium prevents the development of undesirable species, including pathogenic species.

This is why, according to a preferred characteristic, in the method which is the subject of the invention, 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, Schizosaccharomyces or Torulaspora,

- at least one acetic bacterium belonging to the genus Acetobacter, Gluconobacter, or Komagataeibacter.

According to a particularly preferred characteristic of the inventive process, 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, and

- at least one yeast chosen from Saccharomyces cerevisiae, Saccharomyces boulardii, Schizosaccharomyces pombe and Torulaspora delbrueckii.

According to another particularly preferred characteristic of the inventive process, said at least one acetic bacterium originates from an cider vinegar or from an unpasteurized wine vinegar. The particular species involved is not critical, the interesting point being that one of the species present in the original vinegar will grow and establish itself in the consortium alongside lactic acid bacteria and yeast.

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

In an original way, 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 a part of a plant, for example leaves of tea, coffee beans, cocoa pods, or the like. An infusion has the advantage that the plant is briefly subjected to high temperatures, ensuring proper 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 be impacted during the infusion phase and due to 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 culture medium for the preparation of microorganisms is a tea infusion comprising a source of sugars chosen from glucose, fructose 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, with the addition of 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 environment, totally vegetal and without synthetic inputs, ensures the vital comfort of each of the microorganisms. It offers each of them the possibility of meeting 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 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, under the terms of which a composition of microorganisms with a total flora of between 10 ⁵ CFU / g and 10 ⁷ CFU / g. A microbiological enumeration of viable flora indicates that an equilibrium of flora has been created in the environment. The consortium is visualized by the presence of a biofilm on the surface. The composition is maintained as long as desired by transplanting onto new nutrient medium. The composition can also be stored by refrigerating at 4 ° C or by freezing, for later use.

Thus, according to a preferred embodiment of the method which is the subject of the invention, the 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, for obtain a consortium of said microorganisms, comprising a total flora of 10 ⁵ CFU / g to 10 ⁷ CFU / g. 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 special measures to ensure its asepsis with respect to exogenous contaminants, unlike conventional batch culture methods. It can be hypothesized that the choice of microorganisms having 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 O2 gradient of the medium and its redox potential, and also on the appearance of a biofilm produced by microorganisms at the water-air interface. . This biofilm advantageously makes it possible to protect the medium from environmental contaminants, chemical or biological. It materializes the cooperation of the three groups of microorganisms and contributes to the robustness of the process.

The consortium for the preparation of microorganisms described above is capable of reacting with a plant substrate, in an aqueous medium. It is able to direct the extractive fermentation of all kinds of plants, to make accessible, and possibly transform, a great diversity of the compounds they contain. It is expressly specified that the fermentation medium prepared in step a) contains the plant on which the extraction process is applied to obtain an extract of said plant, rich in active compounds. As will be described in detail later, the plant substrate can consist of a whole plant, a part or a fraction of a plant.

In one embodiment of the process for producing a plant extract according to the invention, in step b), the fermentation medium is inoculated at an inoculation rate of between 10 ² CFU / g and 10 ⁴ CFU / g , and incubated at a temperature between 25 ° C and 30 ° C, for 2 to 20 days, and generally between 10 and 15 days.

It appeared that, even inoculated at relatively low levels, 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 microorganisms. This makes it possible to maintain it in the culture medium for several days, or even a few weeks, without human intervention. Although fermentation takes place in principle anaerobically, the process is carried out here in an open environment for gas exchanges with the atmosphere, and without constraint. particular to ensure sterility. In particular, surprisingly, it is observed that no pathogenic flora develops. It is hypothesized that it is the conditions induced and tolerated by the consortium in the reactor which prevent the development of the pathogenic flora present on the substrate and in the environment, even under non-aseptic conditions.

The composition of inoculated microorganisms is able to direct the extractive fermentation of all kinds of plants. The substrate can be chosen from higher plants of any type, whether they are flowering plants, aromatic plants, cereals, fodder plants, protein crops, oilseeds, or others, representing a great diversity. of compounds likely to be used and upgraded. It can also belong to 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 one characteristic of the process 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 very versatile and is of a general, even universal, character. Remarkably, under the defined conditions, the consortium is able to adapt to different types of substrates sensitive to putrefaction (cereals, fodder plants, algae, plants rich in proteins, etc.), rich in minerals or in aromatic compounds (rosemary, lavender, chamomile, ginger, turmeric, coffee, tea, cocoa ...), as well as unicellular organisms such as spirulina for example. 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 can be nettle (Urtica dioica), pastel (Isatis tinctoria), Damascus rose (Rosa damascena), Jujube (Ziziphus zizyphus), Ginkgo biloba , rosemary (Rosmarinus officinalis), lavender (Lavandula), chamomile (Roman chamomile Chamaemelum nobile or wild chamomile, German, matricaria ...), heather (especially the species C alluna 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 may be that which is known in the state of the art as containing compounds of interest or having particular properties, such as turmeric root, the fruit of the Jujube tree, the lavender flower, etc. . We can also choose other parts of the plant, which can reveal their content of active compounds neglected, even ignored until today, but which it is possible to extract and enhance thanks to the synergistic action of the consortium. microbial according to the invention. Vegetable co-products can also be used: the pastel sheets 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 predominant 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 whole plant. a plant part, or as a solid co-product resulting from a treatment of said plant for other purposes and in other processes. 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 between 25 ° C and 90 ° C. ° C, and if necessary an addition of sugar is made, before or after heating, to bring the fermentation medium to a Brix degree of between 2 and 10. The sugar contribution can go 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 obtained from a plant or a part of a fresh or dry plant, in a liquid or essentially liquid form, in particular 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. This concerns fresh fruit juices, flower nectar, as well as all plants in the aqueous phase with or without heating. Fragments can remain dispersed in the liquid phase, such as fruit pulp, the leaves of an infusion, or cells of spirulina. The liquid phase resulting from the extraction of the pastel after precipitation and decantation of the pastel pigment is a co-product which should be mentioned among other equally interesting ones.

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 between 25 ° C and 85 ° C, and if necessary an addition of sugar is carried out before or after heating, to bring the fermentation medium to a Brix degree of between 2 and 10. The sugar contribution can go up to 100 g / l of fermentation medium if necessary. . For example, the liquid substrate supplemented 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 which a person skilled in the art will be able to identify.

In 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 of these. Note that honey can make its own contribution to the properties of the extract obtained. For example, certain honeys that are 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 putrefaction phenomenon is observed during the extraction-fermentation process, while plant substrates are generally loaded with microbial flora causing their degradation, as is particularly nettle. It can be used immediately or stored by a known technique. Anyway, it can undergo different operations before being used or stored, preferably between 4 ° C and 18 ° C.

It is specified that the plant extract obtained can consist of the liquid supernatant alone or else of the whole of the culture medium which can be harvested with the biomass and optionally undergo drying. Indeed, at the end of the extraction-fermentation process, we can focus on the supernatant in which the compounds extracts are dissolved, in which case the process for producing a plant extract according to the invention may comprise:

- a stage of filtration of the fermented extract to remove plant residues,

a sterilizing filtration step to eliminate the microbial flora, commonly performed on a 0.2 μm filter.

These filtrations will be omitted if the biomass is conserved, as is the case when seeking to recover active ingredients that have remained in the biomass, but made bio-available by the 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.

Whether the plant extract is a solute or a mass, we will make sure to stop fermentation and other undesirable processes, in order to stabilize the product obtained. Any known means can 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 lyophilize the aqueous extracts and the solid extracts, dry the solid extracts, for example by passing them in an oven between 30 ° C. and 45 ° C., and grind them more or less finely depending on the intended use.

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

The process which has just been described provides access to compounds which are rarely extracted by conventional solvent-based methods or even by targeted enzymatic methods. This ability is attributed to the fact that the consortium at work offers a great diversity of biochemical tools, some of which are capable of breaking up supramolecular structures of the plant, of simplifying macro-compounds to make them soluble. and thus free them without degrading them. There is thus in a single step, called fermentation-extraction, a fermentation (of the plant substrate) and an extraction (of the intact active ingredients initially present in the plant, towards the liquid fermentation medium). Therefore, the process can be qualified as an all-in-one extraction / fermentation / transformation process. It is emphasized that it constitutes a significant time saving and reduces the operations involving operators. It meets current requirements in terms of eco-responsibility: no synthetic input, open system culture conditions that consume little energy and without asepsis, operating autonomy during the process. It can be industrialized and economically profitable.

A plant extract containing active compounds, obtained by a process 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.

A plant extract produced from a substrate chosen from among flowering plants, aromatic plants, cereals, fodder plants, protein crops, oilseeds, and in particular nettle, pastel, rose may be concerned in particular. from Damascus, 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, physicochemical and organoleptic properties, especially olfactory, with in some 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 plant extract fermented 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 a dermatological product, a cosmetic product, a perfume, a food supplement or a 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 use. 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 an extract of fermented spirulina 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 details of which will appear, in the light of the description which will be given of various variant embodiments, in relation to the appended figures, in which:

FIG. 1 represents the change in the iron content in a nettle extract according to the invention.

Figure 2 shows the change in total acidity in the same nettle extract.

Figure 3 shows the level of live fibroblasts at different concentrations of nettle extract E1. FIG. 4 shows the level of living keratinocytes at different concentrations of the E1 extract.

Figure 5 shows the influence of this nettle extract on different activities.

FIG. 6 shows the level of living fibroblasts at different concentrations of the extract of Ginkgo biloba E3 according to the invention.

FIG. 7 shows the level of living keratinocytes at different concentrations of the same extract of Ginkgo biloba.

Figure 8 shows the influence of this E3 extract on different activities.

FIG. 9 gives the absorption spectra of turmeric extracts according to the invention.

FIG. 10 shows the level of living fibroblasts at different concentrations of the E6 extract (pastel leaves obtained from decoction) according to the invention.

FIG. 11 shows the level of living keratinocytes at different concentrations of the same extract.

FIG. 12 shows the level of living fibroblasts at different concentrations of the E7 extract (juice coproduced from pastel) according to the invention.

FIG. 13 shows the level of living keratinocytes at different concentrations of the same E7 extract.

FIG. 14 shows the level of living fibroblasts at different concentrations of the E8 extract (native pastel sheets) according to the invention.

FIG. 15 shows the level of living keratinocytes at different concentrations of the same E8 extract.

Figure 16 shows the influence of extract E6 on different activities.

Figure 17 shows the influence of the E7 extract on different activities.

Figure 18 shows the influence of the E8 extract on different activities.

FIG. 19 gives the absorption spectra of a spirulina fermentation medium according to the invention.

FIG. 20 represents the effect of extracts according to the invention on plant cells.

EXAMPLE 1: Preparation of a composition of microorganisms

The selected microorganisms are introduced in dry or wet form into sweet 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 minutes to 60 minutes. Commonly, 5 g / l to 10 g / l of tea leaves infused for 15 minutes at 80 ° C are used. The infusion can be used as is with the leaves, or more conveniently the aqueous phase is filtered. Sucrose is then dissolved in the aqueous phase to 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 bacteria are provided by a quantity of unpasteurized vinegar such that the pH of the nutrient medium remains in a range of 4 to 5. The microorganisms develop 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 Brix level have decreased indicating that the sugars are being consumed. Subculture on fresh nutrient medium is then carried out according to conventional techniques known in the state of the art. Compositions of microorganisms have been developed according to this procedure.

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

Composition C2: Composition C2 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 5% cider vinegar. 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 consortium's biofilm is formed from 15 days, although a little less important than in composition C1.

Formation of the consortium: A microbiological count of 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.

By way of 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 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, sweet black tea identical to the nutrient medium of composition C1 was incubated under the same conditions. He developed mold contamination after 5 days. None of the tests showed biofilm formation.

EXAMPLE 2: Preparation of plant extracts

Extract E1

Nettle powder (Urtica dioica) is mixed with dechlorinated water heated to 80 ° C at a level of 50 g / kg of medium. Sucrose is then added in an amount of 50 g / kg of medium and left to cool. Composition C2 prepared according to Example 1 is added in an amount of 1% and the whole is incubated at 28 ° C for 11 days. The nettle extract E1 is obtained, with the residue of nettle powder. 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

Nettle powder (Urtica dioica) is mixed with dechlorinated water heated to 80 ° C at a level of 50 g / kg of medium. The medium is then sweetened up to 50 g / kg of medium and left to cool. Composition C2 prepared according to Example 1 is added in 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 taken and then subjected to a first filtration to remove plant residues and to a second sterilizing filtration over 0.2 µm to remove the ferments. The extract E2 is obtained. Extracts of rosemary (Rosmarinus officinalis), Damascus 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 in an amount of 50 g / kg. The Composition C2 prepared according to Example 1 is added to the aqueous suspension of Ginkgo in an amount of 2%. The whole is incubated for 13 days at 28 ° C. The test supernatant is taken and then filtered to remove plant residues and sterile to remove ferments.

Extract E4

Turmeric (Curcuma longa, root powder) is put at 200 g / kg in 50 g / kg sugar water heated at 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 assay 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 a level of 200 g / kg. Visual observation of the two samples shows a clear difference in behavior: the supernatant of the fermented turmeric extract is yellow and cloudy in color, indicating the solubilization of compounds such as curcumin or other colored molecules, while the turmeric in the The water is translucent with a large 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 the leaves in water at 80 ° C, then sucrose was added at the level of 50 g / l. After cooling to room temperature, composition C1 prepared in Example 1 is added in an amount of 2%. The whole is incubated at 25 ° C for 14 days. The liquid phase acidified to an extent of 2% (total acidity in acetic acid equivalent) is taken and then filtered through a 0.2 μm filter.

Extract E6

The extraction of the pastel blue pigment from the plant of the same name (Isatis tinctoria) requires the treatment of a large quantity of leaves in decoction in water. The pigment yield is low since with 1 ton of fresh leaves, 1 kg of pigment is extracted. The extraction process targeted on pastel therefore generates coproducts in large quantities, which may contain compounds of interest for other applications. Obtaining the pigment uses enzymatic tools derived 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 odors. pestilential during the spreading or storage of this waste. We were interested in three co-products, namely the fresh leaves after decoction in water (extract E6), the liquid phase resulting from the extraction of pastel obtained after precipitation and decantation of the pastel pigment (extract E7) and the juice from the decoction of fresh leaves (extract E8).

Isatis leaves having undergone a first decoction extraction were used to prepare the fermented extract E6. To do this, 8.5 kg of whole wet leaves (containing about 15% dry matter) are first decocted 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 in an amount of 2%. The whole is incubated for 10 days at 28 ° C. The fermentation supernatant is taken and then filtered to remove plant residues, then sterile over 0.2 μm to remove the ferments.

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 3.10 ⁷ CFU / g. The fermentation controlled and orchestrated by the consortium of microorganisms of composition C2 has therefore made it possible to overcome the endogenous flora of the isatis leaves. The minerals released during the fermentation were assayed 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 enhance this co-product, which is currently being discarded, by treating it by fermentation. The juice was heated to 80 ° C and, at this temperature, sucrose is added in an amount of 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 in an amount 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 for 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 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 min at 80 ° C., then sucrose is added at a level of 50 g / l. The sweet juice is maintained at this temperature for 15 minutes. After cooling, composition C2 is added in an amount 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 favorable for 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 vegetal odor, and foaming. The minerals released during the fermentation were assayed 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 is 63.0 mg / kg and that of iron is 15.2 mg / kg. These mineral concentrations are significant and interesting for cosmetic applications. The foaming power of E8 extract can be harnessed for gentle cleansers. It is also noted that the extract E6 obtained from leaves obtained from the production of pastel has made it possible to access quantities of minerals ranging from 50% to 70% of those obtained from native leaves (extract E8). The process according to the invention shows that an enhancement of pastel co-products must be considered.

Extract 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, ...). The extraction of the latter currently requires complex processes using multiple steps, solvents or synthetic adjuvants or energy-intensive processes (ultrasound, microwave, vortex, freezing / thawing). Spirulina extraction-fermentation was performed to release phycocyanin and increase its shelf life in wet form. The spirulina in the form of a dry powder at 20 g / l is placed in dechlorinated water heated to 80 ° C. Sucrose is added in an amount of 50 g / kg. Composition C1 prepared according to Example 1 is added to the aqueous suspension of spirulina in an amount of 2%. The assay is incubated for 6 days at 28 ° C.

Extract E10

Callune 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 which may 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 further proposed to sweeten the fermentation medium with honey, although the acidity of heather and honey, together 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 flowers in buds are introduced at 10 g / l in water and infused for 30 minutes at 80 ° C-90 ° C. The infusion is filtered and when its temperature is below 45 ° C, the chestnut honey is added at a level of 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 level of acid formed is close to 2.5%, which indicates with the presence of a biofilm, that the consortium has grown. The supernatant is filtered through 0.2 μm to ensure its microbiological stabilization. It can be seen that the consortium according to the invention perfectly ensured the extractive fermentation, despite difficult conditions.

EXAMPLE 3: Extraction of iron from a nettle extract

Nettle being 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 nettle extract E1 was compared with two other media prepared as follows:

- Fermentation is carried out in a manner similar to that of E1, but the medium is filtered before adding composition C2 to remove the nettle residues (the fermentation medium is noted ORT-SR).

- 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 from the three media are taken during the fermentation at D0, D3, D6 and D12 and the iron is determined by colorimetry with potassium permanganate. The results obtained are presented in FIG. 1. The samples with ORT-AR residues exhibit an increased rate of extracted iron over time, whereas this rate does not change in the medium without ORT-SR nettle residues. This attests to the continuous extraction of iron from the residues of the plant during the fermentation, the maximum extraction rate being achieved from the 6 ^th day by the action of added enzymes. In fact, iron extraction is also visible in the samples without added ferment ORT-SSF, but it is slower since it does not reach its maximum until after 12 days. On the other hand, the ORT-SSF samples show a significant proliferation of the native flora of the nettle, 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 powerful and characteristic vegetal odor. The total acidity was also measured by colorimetric determination with sodium hydroxide in the presence of phenolphthalein. It is expressed in% of acetic acid equivalents, as presented in FIG. 2. The sugar level in degrees Brix was recorded at D0 and D6.

The difference in acidity between the three environments is marked from the 6 ^th day and continues until the ^11th day. In the ORT-SSF test, acidity stabilizes from the ^8th day indicating that acid production is stopped, thus signing the absence of fermentation itself, 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, 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 fermentation is characteristic of the establishment of the composition C2 consortium in 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 wild microbial development, protecting both the liquid extract and the residual biomass of the plant as well. processed.

EXAMPLE 4: Biological properties of a nettle extract

Cytotoxicity

The nettle extract E2 obtained in Example 2 was tested with regard to its cytotoxicity in vitro 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 diluted in cascade beforehand 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 FIG. 3 (fibroblasts) and in FIG. 4 (keratinocytes), show that the E2 extract shows no cytotoxicity compared to the control without extract, on the two cell types, when it is present at a level between 0.01% and 1% in vitro, which is compatible with cutaneous applications.

Cellular activities

The E2 extract was then tested on a genomic chip to identify the stimulated activities. The extract is diluted beforehand in culture medium, to a non-toxic concentration of 0.01%, then incubated with normal human epidermal keratinocytes for 24 hours. The messenger RNAs (mRNA) are extracted and then analyzed by RT-qPCR (Real Time quantitative Polymerase Chain Reaction). The results are shown in Figure 5. They are expressed in%, positive in the event of overexpression and negative in the event of inhibition relative 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 extract of Ginkgo biloba E3 obtained in Example 2 was tested with regard to its cytotoxicity in vitro on two normal human cell types: keratinocytes and fibroblasts, according to the procedure identical to Example 4. The results given to Figure 6 (fibroblasts) and Figure 7 (keratinocytes), show that the E3 extract shows no 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 skin applications. Cellular activities The E3 extract was then tested on a genomic chip to identify the stimulated activities, after dilution in culture medium, at a non-toxic concentration of 0.01%, then incubation with normal human epidermal keratinocytes for 24 hours.

The messenger RNAs (mRNA) are extracted and then analyzed as above. The results are presented in FIG. 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 changes in the expression of these genes are of interest for cosmetic applications aimed at strengthening the barrier function of the skin.

EXAMPLE 6 Extraction of curcumin and derivatives of an extract of Curcuma longa

We know that turmeric is naturally rich in curcuminoids, mainly curcumin. The extract E4 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 oven dried at 45 ° C. The liquid phase (turmeric-F-juice) and the dried solid phase (turmeric-F-powder) are analyzed spectrophotometrically and compared to the native unfermented turmeric taken up in water (turmeric-FLO). To this end, the turmeric powders, fermented and native, are taken at 100 mg / ml and ¼ dilutions are made in order to be able to compare the samples. The samples are centrifuged and the supernatant is analyzed by visible spectrophotometry. The spectra obtained are presented 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). The curcumin content appears to be equivalent in fermented turmeric supernatant (turmeric-F-juice) and native turmeric (turmeric-FhO) showing that fermentation did not degrade this compound. In addition, fermented turmeric powder (turmeric-F-powder) exhibits very marked curcumin peaks and metabolizing compounds, about four times greater than that 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 form of bioavailable and water-extractable curcumin. EXAMPLE 7: Biological properties of pastel extracts and pastel co-products

Isatis tinctoria, known for the production of pastels, contains other compounds which may exhibit properties of interest in cosmetics. 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 extracts E6, E7 and E8 were tested with regard to their cytotoxicity in vitro on human keratinocytes and fibroblasts, according to the procedure described in Example 4, with extract concentrations of between 0.01% and 5%. The results relating to the extract E6 (fermented isatis leaves, denoted FISAF) are given in FIG. 10 (fibroblasts) and in FIG. 11 (keratinocytes). The results relating to the extract E7 (fermented isatis juice, noted JISAF) are given in FIG. 12 (fibroblasts) and in FIG. 13 (keratinocytes). The results relating to the extract E8 (untreated leaves, denoted DECISA) are given in FIG. 14 (fibroblasts) and in FIG. 15 (keratinocytes). They show that the E6 extract does not show any 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%. Skin applications can therefore be considered.

Cellular activities

The extracts E6, E7 and E8 were then tested on a genomic 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 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, involucrine, occludin, Serine Protease I Nhibitor Kazal-type 5 or SPINK5). The overexpression of the gene corresponding to the peptidase-3 inhibitor is particularly high at a level of about 450%. Concerning the E8 extract, several genes are overexpressed between 80% and 180%. The overexpression of the decorin and the involucrin around 250% is particularly high. It can be said 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 an Extract of Spirulina

The extract E9 prepared in Example 2 was analyzed. On days D0, D3 and D6, an aliquot of spirulina is taken 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 peaks characteristic of spirulina, around 460 nm for carotenoids and around 600. nm for phycocyanin. The biofilm also contains phycocyanin.

On the other hand, we know that spirulina is sensitive to microbial contamination in aqueous media due to its rich nutrient composition and 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 exhibits wild contamination, identified by the foul odor of putrefaction. On the contrary, in the test carried out with composition C1, the unpleasant marine odor disappeared. It can therefore be seen that the process according to the invention made it possible to avoid wild fermentation of the medium. Thus, the extraction / fermentation makes it possible to extract compounds of interest from spirulina and also makes it possible to hygienize the spirulina in the aqueous phase. Therefore, it is possible to keep it without adding a preservative. It can be used as a source of assets.

EXAMPLE 9: Care cream comprising a nettle extract

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

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 the E2 extract of fermented nettle (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 coconut caprylate (0.75 g), coconut butter (0.5 g) and jojoba oil (3.75 g). Under strong agitation with the deflocculating paddle. 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 of between 5 and 5.5 with 1N sodium hydroxide and to a mass of 50 g with demineralized water. The resulting cream is smooth, slightly shiny and moderately compact. The composition comprising 10% E2 extract is very slightly off-white with a slight odor characteristic 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 the blood microcirculation. The activities present in the extract E3 demonstrated in Example 4 are advantageous for use on the skin. The E3 extract was incorporated without difficulty into a skin care cream and into a gel.

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 copolymer of hydroxyethyl acrylate and sodium acryloyldimethyl taurate ) is added with stirring. From 0.5 g to 5 g of fermented Ginkgo biloba extract E3 (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 slowly added to the aqueous phase, as well as 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 demineralized water. The cream obtained, comprising 10% 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 feel and 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. 0.5 g to 5 g of fermented Ginkgo biloba extract E3 (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 mixture obtained is adjusted to a pH of 5 to 5.5 if necessary, and by mass to 50 g with demineralized water. The gel obtained has a very gelled texture which spreads well on the skin.

EXAMPLE 11: Anti-itch lotion

The extract E10 made from 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 people with pimples in several parts of the body, signs of an inflammatory reaction following mosquito bites. The people treated affirmed that they no longer felt an itch until 3 minutes to 5 minutes after application. At the end of 24 hours, the buttons 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 consist 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 perfumes, which makes it possible to enhance the fragrant 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 perfume solutions has a very interesting antioxidant potential, making it possible to consider their use in perfumed body care 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 extracts of nettle, rosemary, damask rose and jujube leaves were fermented according to the protocol used for the extract E2 of example 2. The extract of black tea E5 prepared according to example 2 is also employed.

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

Perfume 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, rosemary extract (very fragrant) was added in an amount of 20%. In composition B2, 20% fermented tea (little odorant 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 B1 and B2 fragrances obtained are invigorated by the presence of fermented extracts and the acidity brings volatility to the aromas, 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 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 placed, lesion 2 is left as it is. A control consists of depositing a drop of the E5 extract on a part of the leaf which is healthy and 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 lesion area 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 from 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, true 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 in plant cells and facilitates cellular exchanges with their environment, like a blue light would do on a plant, thus accelerating cell growth. This E5 extract promotes tissue receptivity while maintaining a healthy ground.

EXAMPLE 15: Action on the hydration of plant cells

It is known that the action of salt water on animal or plant tissue causes dehydration of cells due to the difference in osmotic pressure between the outside and 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.

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

The two extracts of nettle E2 and tea E5 were tested on a Peruvian Oca tuber (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 about 40% humidity with 5 ml of liquid at the bottom of the tube, namely respectively: 0.9% NaCl salt water (ES tube), extract of fermented tea E5 1% in salt water NaCl 0.9% (tube Tea-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 become dehydrated. It is observed after 30 days that the tuber of the Ort-F-1% tube exhibits a growth of the order of 2.5 cm in length with the appearance of foliage, while the leaf stems of the Thé-F-1% tubes and ES stretched out very little. It is further noted that the tubercle of the ES tube exhibits mold, which is not the case with Ort-F-1% and Thé-F-1% tubes. However, in the latter tube, the tuber did not show any foliage 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

1 A process for producing a plant extract, characterized in that ^r \\ comprises the steps of: a) - preparing a fermentation medium containing a plant substrate in aqueous phase, b) - inoculating said fermentation medium with a preparation of microorganisms forming a consortium in a sweet 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 bacteria, c) - incubate in a 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 A method of producing a plant extract according to the preceding claim, characterized in that said preparation of microorganisms comprises:

- at least one acetic bacterium belonging to the genus Acetobacter, Gluconobacter or Komagataeibacter.

3 A method of producing a plant extract according to claim 2, characterized in that said preparation of microorganisms comprises:

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

5 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 is 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 is an infusion of tea comprising a source of sugars chosen from glucose, fructose or a mixture of these.

7 -. Process for producing a plant extract according to one of the preceding claims, characterized in that the 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 10 ⁵ CFU / g to 10 ⁷ CFU / 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 10 ² CFU / g and 10 ⁴ CFU / g, and incubated at a temperature 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 the production of a plant extract according to one of the preceding claims, characterized in that the plant substrate is chosen from nettle, pastel, Damascus rose, jujube, Ginkgo biloba, rosemary, lavender , chamomile, heather, ginger, turmeric, coffee, tea, cocoa, spirulina.

11 - Process for the production of 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 from a 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 and then brought to a temperature between 25 ° C and 90 ° C, and if necessary an addition of sugar is carried out, before or after heating, to bring the fermentation medium to a Brix degree of between 2 and 10.

13 A method of producing a plant extract according to one of claims 1 to 9, characterized in that the plant substrate is obtained from a plant or a part of a fresh or dry 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 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 heating, to bring the fermentation medium to a Brix degree of between 2 and 10.

15 - Process for the production of 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 of these.

16 -. Process for the production of 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 remove residues plants, sterilizing filtration to eliminate microbial flora, microbiological decontamination, lyophilization, 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, pastel, 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 obtainable by a process according to one of claims 1 to 16, for the manufacture of a pharmaceutical product, a cosmetic product, d 'a perfume, a dietary supplement or plant treatment product.

20 Use according to the preceding claim for the manufacture of a pharmaceutical product or a cosmetic product further 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.