Use of a Crocus sativus-based composition as a prebiotic

Abstract

The present invention relates to the use of a composition based on crocus sativus as a prebiotic in humans or animals, in particular to increase the quantity of bacteria of the genus Akkermansia in the intestinal microbiota.

Description

Title of the invention: Use of a composition based on Crocus sativus as a prebiotic Technical field

[0001] The invention relates to the use of a composition based on crocus sativus as a prebiotic in humans or animals, in particular to increase the quantity of bacteria of the genus Akkermansia in the intestinal microbiota.

[0002] State of the art

[0003] Our intestinal microbiota is made up of a set of non-pathogenic bacteria, viruses, parasites and fungi, or 1012 to 1014 microorganisms present in the digestive tract of each individual, which represents 2 to 10 times more than the number of cells present in our body. Therefore, its role has been increasingly studied in recent years. Also, the importance of the role played by the intestinal microbiota on the health of the host has been highlighted.

[0004] In this context, inverse correlations have been highlighted between the abundance of the Akkermansia spp bacterial population within the intestinal microbiota and various metabolic (insulin resistance, obesity, etc.) or digestive (irritable bowel syndrome, celiac disease, etc.) disorders. Bacteria of the genus Akkermansia, such as the species A kkermansia muciniphila, generally represent 3-5% of the human microbiota (Belzer & de Vos, 2012 10.1038 / ismej.2012.6; Derrien et al., 2004 doi: 10.1099 / ijs.0.02873-0 / They act in particular on inflammation, by reducing intestinal permeability, which has the consequence of reducing the phenomenon of endotoxemia, at the origin of the production of reactive oxygen species and pro-inflammatory cytokines (Hills RD Jr, Pontefract BA, Mishcon HR, Black CA, Sutton SC, Theberge CR. Gut Microbiome: Profound Implications for Diet and Disease. Nutrients. 2019 Jul 16;11(7):1613) doi: 10.3390 / nul 1071613).

[0005] Recently, it has been shown that a daily intake of Akkermansia muciniphila bacteria in aged mice significantly improved several behavioral responses such as coordination, balance, exploration abilities, anxiety-like behaviors as well as immune functions and oxidative stress parameters (Cerro, E.DD., Larnbea, M., Félix, J. et al. Daily ingestion of Akkermansia mucciniphila for one month promotes healthy aging and increases lifespan in oldfemale mice. Biogerontology (2021), doi: 10.1007 / s10522-021 -09943-w ).

[0006] On the other hand, work carried out on different animal models suggests that an increase in the population of Akkermansia within the intestinal microbiota would prevent the onset of cognitive deficits (Yang Y, Zhong Z, Wang B, XiaX, Yao W, HuangL, Wang Y, Ding W. Early-life high-fat diet-induced obesity programs hippocampal development and cognitive functions via regulation of gut commensal Akkermansia muciniphila. Neuropsychopharmacology . 2019 Nov; 44(12):2054-2064, doi: 10.1038 / s41386-019-0437-l).

[0007] Finally, preclinical data seem to indicate that the presence of the species Akkermansia muciniphila within the intestinal microbiota plays an important role in the gut-brain axis by intervening in particular in the signaling pathway and metabolism of serotonin and consequently, in disorders linked to menopause and premenstrual syndrome (Yaghoubfar R, Behrouzi A, Ashrafian F, et al. Modulation of serotonin signaling / metabolism by Akkermansia muciniphila and its extracellular vesicles through the gut-brain axis in mice. Sci Rep. 2020; 10(1):22119. Published 2020 Dec 17. doi: 10.1038 / s41598-020-79171-8).

[0008] Also, the presence of the species Akkermansia muciniphila within the intestinal microbiota in humans or animals is a sign of good health.

[0009] There is therefore a significant need for a product capable of increasing the Akkermansia population within the intestinal microbiota, in particular to increase the quantity of bacteria of the species Akkermansia muciniphila, preferentially for subjects presenting an intestinal microbiota depleted in bacteria of the genus Akkermansia.

[0010] During their work, the inventors surprisingly demonstrated that the addition of a saffron-based preparation, in particular based on crocus sativus, makes it possible to increase the quantity of bacteria of the genus Akkermansia in the intestinal microbiota and thus act as a prebiotic.

[0011] The health benefits of saffron, particularly Crocus sativus, are well described. For example, its interest in the treatment of depression, but also in healthy populations with mood disorders (Jackson, 2021; doi:10.3389 / fnut.2020.606124), or in the management of stress and anxiety. It has also been shown that chronic stress can cause deleterious effects on health decades later, for example an increased risk of dementia (Wheelan, 2018; doi:10.1093 / brain / awx341).

[0012] Other clinical studies have also shown that saffron supplementation in healthy subjects or in patients with cognitive decline or Alzheimer's disease improved or maintained cognitive performance compared to a placebo, or with an efficacy equivalent to that of reference drugs (Avgerinos, 2020, doi: 10.1007 / s 10072-020-04427-0).

[0013] Clinical studies also suggest that saffron supplementation could reduce inflammatory disorders, both at the digestive level (Tadyon Najafabadi, Borna et al. “Therapeutic effects of saffron (Crocus sativus) versus fluoxetine on Irritable Bowel Syndrome: A double-blind randomized clinical trial.” Advances in integrative medicine 6 (2019): 167-173.), as well as at the joint level (Hamidi Z, Aryaeian N, Abolghasemi J, Shirani F, Hadidi M, Fallah S, Moradi N. The effect of saffron supplement on clinical outcomes and metabolic profiles in patients with active rheumatoid arthritis: A randomized, double-blind, placebo-controlled clinical trial.

[0014] Given its health benefits, many saffron-based preparations are known from the prior art, including preparations based on Crocus sativus extract whose safranal content is high and measured by HPLC. For example, an extract titrated to 0.03% safranal is marketed by the company Pharmactive (as described in WO 2017 / 182688), but also by the company Activ'Inside, titrated to at least 0.2% safranal (as described in FR 3054443).

[0015] Although many studies have highlighted the health benefits of saffron, none describe the use of saffron or crocus sativus extract as a prebiotic, i.e. capable of selectively promoting the growth of certain bacteria or the activity of the microbiota.

[0016] Certainly, it has been shown that certain ingredients can modulate the population of Akkermansia muciniphila within the intestinal microbiota, however with variable results depending on the ingredients but also depending on the populations. For example, resveratrol supplementation has been shown to be effective in Caucasian Americans, but not in other ethnic groups. A pomegranate extract, for its part, was found to have very variable efficacy (with a factor of almost 50) in healthy subjects, depending on whether or not they were producers of urolithin A. Finally, supplementation with kiwi polyphenols was found to be totally ineffective (Verhoog S, Taneri PE, Roa Diaz ZM, Marques-Vidal P, Troup JP, Bally L, Franco OH, Glisic M, Muka T. Dietary Factors and Modulation of Bacteria Strains of Akkermansia muciniphila and Faecalibacterium prausnitzii: A Systematic Review. Nutrients. 2019 Jul 11;11(7):1565; doi: 10.3390 / nul 1071565).

[0017] Thus, to date, it is, on the one hand, impossible to predict the possible effects that an ingredient will have on the population of Akkermansia muciniphila within the microbiota and, on the other hand, no prebiotic effect of saffron has been reported. There is therefore a need for new prebiotic compositions that can overcome its drawbacks and that are capable of acting as a prebiotic, in particular to increase the bacterial population of the genus Akkermansia within the intestinal microbiota. Summary of the invention

[0018] To answer this, the inventors demonstrated that the use of a composition comprising at least one extract of Crocus sativus could be used as a prebiotic composition in humans or animals.

[0019] Indeed, the inventors have discovered that the oral administration of a preparation based on Crocus sativus to rodents, in a model of inflammation, is capable of increasing the bacterial population of the genus Akkermansia within the intestinal microbiota of the animals.

[0020] Also, the invention relates to the use of a composition comprising at least one extract of Crocus sativus as a prebiotic composition. Preferably, the use of the composition comprising at least one extract of Crocus sativus is capable of increasing the population of bacteria of the genus Akkermansia, and more particularly of the species A kkermansia muciniphila, within the intestinal microbiota of a subject, in particular of humans or animals.

[0021] The composition based on Crocus sativus is preferably intended for subjects presenting an inflammatory profile or likely to present an inflammatory profile or an inflammatory syndrome in order to reestablish a healthy intestinal microbiota.

[0022] Advantageously, the composition is capable of increasing the quantity of bacteria of the genus Akkermansia by at least 20%, compared to the quantity of bacteria initially present in the intestinal microbiota of the subject. Such an intake makes it possible to significantly increase the quantity of bacteria of the genus Akkermansia within the intestinal microbiota and thus to reestablish a healthy intestinal microbiota.

[0023] According to another object, the composition is capable of increasing the quantity of bacteria of the genus Akkermansiad, at least one logarithmic unit (1 Log) within the intestinal microbiota, in particular compared to the quantity of bacteria initially present in the intestinal microbiota of the subject.

[0024] In the context of the invention, the saffron extract, in particular from crocus sativus, can be obtained from a ground material of the whole plant, from a part of the plant, ground or not, for example the stigmas, the petals, the bulb, etc. Finally, the extract can also be obtained from an extraction process based, for example, on a maceration of the plant material, or on an aqueous, hydroalcoholic, alcoholic, etc. extraction, accompanied by a heat treatment or not.

[0025] According to a preferred subject, the extract of Crocus sativus constituting the composition and intended to be used as a prebiotic, comprises at least 0.01% of safranal, preferably at least 0.03% of safranal, more preferably at least 0.1% of safranal, even more preferably at least 0.2% of safranal, by weight relative to the total weight of the dry matter of the extract, measured by HPLC.

[0026] According to another preferred subject, the Crocus sativus extract comprises a mixture of molecules comprising: - at least 0.01% of safranal, preferably at least 0.03% of safranal, more preferably 0.2% of safranal, by weight relative to the total weight of the dry matter of the extract, measured by HPLC, and / or - at least 1% crocins by weight relative to the total weight of the dry matter of the extract, measured by HPLC method, and / or - at least 500 ppm of kaempferol-derived flavonoids by weight of dry matter of the extract, measured by HPLC method, and / or - at least 0.5% picrocrocin by weight of dry matter of the extract, measured by HPLC method, and / or - a bulking agent chosen from maltodextrin, sugar, silica and gum arabic, intended to encapsulate said Crocus sativus extract.

[0027] Such a crocus sativus extract may be obtained by any extraction method known from the prior art. Also, the crocus sativus extract is capable of being obtained by the method comprising the following steps: a. Crushing of Crocus sativus containing safranal, b. Aqueous or hydroalcoholic extraction or with an organic solvent, c. Drying of the extract on a bulking agent or mixing with a bulking agent.

[0028] Preferably, step c) of drying or mixing is replaced by a step of impregnation and encapsulation of the extract in the bulking agent in the extraction solution, followed by a heat treatment step, preferably for at least 2 hours at a temperature between 30°C and 95°C.

[0029] According to another aspect of the invention, the composition may comprise at least one extract of Crocus sativus (i) and at least one bacterium belonging to the genus Akkermansia (ii), in particular said composition is a prebiotic composition.

[0030] Preferably, the bacterium belongs to the species Akkermansia muciniphila. Advantageously, the effect of saffron on the population of Akkermansia muciniphila within the microbiota is further improved by the addition of at least one such bacterium of the species Akkermansia muciniphila and makes it possible, for example, to restore a healthy intestinal microbiota more quickly and with greater efficiency, in particular when the intestinal microbiota of the subject is particularly depleted in bacteria of the genus Akkermansia.

[0031] According to an object of the invention, the bacteria present in the composition according to the invention is preferably in pasteurized form. By way of example, a pasteurized form & Akkermansia muciniphila is described in patent EP3347030.

[0032] In order to further optimize the prebiotic effect, the composition according to the invention may preferably comprise at least one additional prebiotic ingredient and / or at least one probiotic bacteria and / or yeast and / or at least one vitamin and / or at least one mineral.

[0033] Finally, the composition is intended for various applications. Also, the composition can be administered orally as a food product, food supplement, beverage, cosmetic product or medicine.

[0034] The composition may also be presented in different galenic forms, such as in the form of a tablet, capsule, stick, patch, cream or gel.

[0035] Preferably, the composition is encapsulated in a food carrier. Once encapsulated, the composition may be in the form of a powder, capsule, tablet, capsule, solution, suspension, emulsion or chewing gum.

[0036] According to a final aspect, the composition according to the invention comprising at least one extract of Crocus sativus (i) and at least one bacterium belonging to the genus Akkermansia (ii) is intended to be used as a prebiotic composition.

[0037] Also, according to a preferred object, the invention also aims at the use of the composition comprising at least one extract of crocus sativus and at least one bacterium of the genus Akkermansia, according to any of the embodiments described above, as a prebiotic. Preferably, its use for increasing the population of bacteria of the genus Akkermansia, and more particularly for increasing the population of bacteria of the species A kkermansia muciniphila, within the intestinal microbiota of a subject, in particular of humans or animals.

[0038] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow. Brief description of the Figures

[0039] [Fig. 1] represents the beta bacterial diversity based on the Bray-Curtis dissimilarity index, on 3 groups of 6 C57 / B16 mice (SHAM, LPS and LPS-SAF group).

[0040] [Fig.2] is a table representing the results of the microbiota analysis on the 3 groups of 6 C57 / B16 mice (SHAM, LPS and LPS-SAF group) by the LEfSe method. The parameters used for the analysis were a logarithmic ADL score >2 and a threshold for the adjusted p value of 0.2.

[0041] [Fig.3] represents the abundance of bacteria belonging to the genus Akkermansia on the 3 groups of 6 C57 / B16 mice (SHAM, LPS and LPS-SAF groups). The value of each individual as well as the median and the interquartile range are represented. **p<0.01. Detailed description of the invention

[0042] Definition

[0043] By "increasing the quantity of bacteria" within the meaning of the invention, it is meant that after administration of the composition according to the invention, the quantity of bacteria of the genus Akkermansia is higher in the intestinal microbiota than before administration.

[0044] By "Crocus sativus extract" or "saffron extract" within the meaning of the invention, is meant at least one molecule or a set of several molecules derived from all or part of Crocus sativus containing crocins and / or safranal. It may be a specific selection of native molecules present in Crocus sativus or molecules obtained by any type of transformation of said native molecules. The raw material used to obtain the extract may consist of all or part of Crocus sativus containing crocins and / or safranal, in particular the Crocus sativus extract may be obtained preferentially from the stigmas and / or petals of Crocus sativus, whether crushed or not. More preferably, the extract is obtained by the process described in patent FR 3054443, incorporated by reference.

[0045] By "initially present in the intestinal microbiota" within the meaning of the invention, we mean the quantity of bacteria present in the microbiota before administration of the composition according to the invention.

[0046] For the purposes of the invention, the term "total weight of dry matter of XXX" means the dry matter obtained when the water is removed. It can be obtained in particular by loss on drying at 105°C. The dry product obtained can be in solid or liquid form.

[0047] For the purposes of the invention, the term "prebiotic" means a food ingredient capable of inducing an increase in the diversity of the intestinal microbiota, in particular a partially non-digestible food ingredient capable of selectively stimulating, in the digestive system, the multiplication or activity of one or a limited number of bacterial groups capable of improving the physiology or health of the host.

[0048] For the purposes of the invention, the term "inflammatory profile" means a subject exhibiting inflammation, i.e. at least one reaction, in particular several reactions which occur in the body irritated by an attack, in particular by a pathogenic agent or considered pathogenic by the body. Preferably, the inflammation is located in the digestive system.

[0049] For the purposes of the invention, the term "subject" means a human or an animal. Said subject may present an inflammatory profile or is likely to present, preferably in the short, medium or long term, an inflammatory profile, or an inflammatory syndrome in the digestive system.

[0050] By "logarithmic unit" of a bacterial population, we mean the decimal logarithm (LoglO) of the number of CFU (Colony Forming Unit) per gram of stool.

[0051] Use of the composition according to the invention

[0052] The present invention therefore relates to a first composition comprising at least one extract of Crocus sativus, said composition is used as a prebiotic composition.

[0053] Also, according to a first aspect, the invention relates to the use of a composition comprising at least one extract of Crocus sativus as a prebiotic composition, said composition being preferentially administered to a subject, said subject being chosen from humans and animals.

[0054] According to a preferred subject, the invention relates to the use of such a composition based on an extract of crocus sativus, as a prebiotic composition capable of increasing the quantity of bacteria of the genus Akkermansia in the intestinal microbiota of a subject.

[0055] The human or animal subject may present or be likely to present an inflammatory profile or an inflammatory syndrome or be healthy, that is to say that the intestine does not present signs of inflammation, preferentially, the subject presents an inflammatory profile or an inflammatory syndrome.

[0056] The composition according to the invention aims to increase the quantity of bacteria of the genus Akkermansia in the intestinal microbiota of a subject, such a bacterial population being associated with signs of good health for the host. Also, preferably, the invention aims at the use of the composition according to the invention as a prebiotic composition capable of increasing the quantity of bacteria of the genus Akkermansia by at least 20%, relative to the quantity of bacteria initially present in the intestinal microbiota of the subject. More preferably, the composition according to the invention is capable of increasing the quantity of bacteria by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0057] Such a quantity is particularly advantageous for restoring a healthy intestinal microbiota and better bacterial diversity, a sign of good health for the host.

[0058] According to another object, the composition makes it possible to increase the quantity of bacteria of the genus Akkermansiad, at least one logarithmic unit (1 Log) within the intestinal microbiota.

[0059] Preferably, the composition according to the invention intended to be used as a prebiotic, preferentially in order to promote Akkermansia spp. within the microbiota, comprises at least one extract of crocus sativus.

[0060] The crocus sativus extract can be obtained from the whole plant, crushed or not, or from a part of the raw material, crushed or not, for example from the stigmas. In the context of the invention, the crocus sativus extract is preferably obtained from the stigmas and / or petals of Crocus sativus, in particular in order to obtain a high safranal content.

[0061] The stigma(s) and / or petal(s) of Crocus sativus constitute(s) a part of the raw material as such, optionally dried and / or ground. Thus, the stigma(s) and / or petal(s) of Crocus sativus can be incorporated into the composition according to the invention in fresh form, that is to say directly after harvesting without treatment, or in dry form or dry and ground form.

[0062] The Crocus sativus extract comprises a mixture of molecules, preferably at least safranal and / or crocins, and the safranal is present at a level of at least 0.01%, preferably at least 0.03%, more preferably the extract comprises at least 0.1% safranal, even more preferably at least 0.2% safranal, by weight of dry matter relative to the Crocus sativus extract, measured by HPLC (High Performance Liquid Chromatography) method.

[0063] Said extract can be obtained by any extraction method well known to those skilled in the art making it possible to obtain an extract comprising safranal and crocins.

[0064] According to a preferred subject of the invention, the extract also comprises other molecules chosen from flavonoids derived from kaempferol, and picrocrocins.

[0065] Preferably, the Crocus sativus extract comprises: - at least 0.01% of safranal, preferably at least 0.03% of safranal, more preferably at least 0.2% of safranal by weight relative to the total weight of the dry matter of the extract, measured by HPLC, and / or - at least 1% crocins by weight relative to the total weight of the dry matter of the extract, measured by HPLC method, and / or - at least 500 ppm of kaempferol-derived flavonoids by weight of dry matter of the extract, measured by HPLC method, and / or - at least 0.5% picrocrocin by weight of dry matter of the extract, measured by HPLC method, and / or - a bulking agent chosen from maltodextrin, sugar, silica and gum arabic, intended to encapsulate said Crocus sativus extract.

[0066] The extract can also be obtained by implementing a method comprising the following steps: - possibly drying, - crushing of Crocus sativus containing safranal, preferably between 50 and 500 pm, - aqueous or hydroalcoholic extraction or with an organic solvent, - drying the extract on a bulking agent or mixing with a bulking agent, or - impregnation and encapsulation in the bulking agent in the extraction solution, and drying, and / or heat treatment.

[0067] Preferably, the extract is also capable of being obtained by implementing a method comprising the following steps: - possibly drying, - crushing of Crocus sativus containing safranal, preferably between 50 and 500 pm, - aqueous or hydroalcoholic extraction or with an organic solvent, - impregnation and encapsulation in the bulking agent in the extraction solution, and - heat treatment, preferably for at least 2 hours at a temperature between 30°C and 95°C.

[0068] The bulking agent may be among dextrins, maltodextrin, sugar, silica, gum arabic, glycerin, fats, vegetable waxes, hydrogenated or non-hydrogenated vegetable oils, proteins, peptides, alginates, phospholipids, starch, polyols (for example sorbitol or maltitol, cellulose, calcium or magnesium carbonate, or gum arabic, more preferably maltodextrin.

[0069] Said extract also preferably underwent a heat treatment after impregnation and encapsulation for at least 2 hours, preferably between 24 hours and 96 hours; at a temperature between 30°C and 95°C, preferably at a temperature between 30°C and 60°C.

[0070] The heat treatment step can optionally be carried out at any time during this process, such as: - Between the drying and grinding stage, or - Between the grinding and extraction stage, or - Between extraction and the impregnation and encapsulation step, or - preferably after the impregnation and encapsulation step.

[0071] When the extract is impregnated in a bulking agent, the percentages of molecules present in the extract are given in weight of dry matter of the extract including the bulking agent.

[0072] The grinding can be carried out by any known suitable means, in particular by a knife, pin or hammer mill, preferably a pin mill.

[0073] The extraction step can be carried out by any known suitable means. In the case of aqueous extraction, the ground material is introduced into the water at a rate of 50g / L.

[0074] In the case of a hydroalcoholic extraction, the solvent may in particular be ethanol, preferably 60% v / v ethanol. The ground material is introduced into the hydroalcoholic solution at a rate of 50g / L.

[0075] In the case of extraction with an organic solvent, the solvent may in particular be methanol or ethyl acetate, preferably methanol at 30% v / v. The ground material is introduced into the organic solvent at a rate of 100g / L.

[0076] After extraction, the process may also include an acidification step. This step consists of adding acid to the aqueous or hydroalcoholic solvent. It makes it possible to reduce the pH of the extraction solution to between 3 and 5. It may in particular be carried out under the following conditions: addition of citric acid or hydrochloric acid to the hydroalcoholic solvent to adjust the pH to 4.

[0077] The step of impregnation and encapsulation simultaneously on the support consists of adding a filler agent to the extraction solution, i.e. in the liquid state.

[0078] More preferably, such a step is also carried out simultaneously with the extraction step, which makes it possible to trap the safranal molecules on the one hand and the crocins on the other hand during the extraction. The trapping of the safranal molecules results in obtaining a highly concentrated safranal extract of at least 0.2% and encapsulated, allowing better stability of both the safranal and the crocins. This step consists of high-speed stirring of the extraction solution containing the bulking agent, the excipient or the support.

[0079] More preferably, the Crocus sativus extract is an extract obtained according to the process described in patent FR 3054443, incorporated by reference.

[0080] According to a final object, the composition according to the invention is preferentially delivered at a dose making it possible to provide humans or animals daily with at least 0.2 mg / kg of body weight of a composition comprising at least one extract of Crocus sativus.

[0081] Composition according to the invention

[0082] According to another aspect, the invention relates to a composition based on a combination of two active ingredients, at least one extract of Crocus sativus (i) and at least one bacterium belonging to the genus Akkermansia (ii).

[0083] Thus, the composition comprises at least one extract of Crocus sativus (i) and at least one bacterium belonging to the genus Akkermansia (ii). Preferably, the bacterium belongs to the species Akkermansia muciniphila.

[0084] The bacteria contained in the composition according to the invention may be inactivated, attenuated or live. Preferably the bacteria is in pasteurized form.

[0085] According to a preferred object, the composition comprises between IxlO4 and IxlO12 CFU of bacteria, more preferably between IxlO5 and IxlO11 and even more preferably between IxlO6 and IxlO10 CFU of bacteria of the species Akkermansia muciniphila.

[0086] Also, the effect of saffron on the Akkermansia population within the microbiota is further enhanced by the addition of at least one such bacterium Akkermansia muciniphila , in particular when the composition comprises at least IxlO6 CFU, and allows for example to restore a healthy intestinal microbiota more quickly and with better efficiency, in particular when the intestinal microbiota of the subject is particularly depleted in Akkermansia.

[0087] To further improve the effect of the composition according to the invention, it may also comprise at least one additional prebiotic ingredient and / or at least one probiotic bacteria and / or yeast, and / or a vitamin and / or a mineral.

[0088] When the composition comprises an additional prebiotic ingredient, this is preferentially chosen from rinulin, FOS (Fructooligosaccharides), GOS (Galactooligosaccharides), XOS (xylooligosaccharide) or MOS (Mannanoligosaccharide).

[0089] When the composition comprises a probiotic bacterium and / or yeast, these are chosen from bacteria and / or yeast of the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, Saccharomyces, Enterococcus, Pediococcus and Escherichia.

[0090] When the composition comprises a vitamin, this is chosen from vitamin A, C, D, E, K, B1, B2, B3, B5, B6, B8 and B12.

[0091] When the composition comprises a mineral, the latter is chosen from calcium salts, zinc salts, magnesium salts and selenium salts.

[0092] According to another object of the invention, the composition can be encapsulated or microencapsulated in a food support chosen from a maltodextrin, a gum arabic, a hydrogenated oil, a non-hydrogenated oil, a wax, an alginate, starch and a protein.

[0093] The composition according to the invention may be in any form suitable for the uses according to the present invention. Preferably, the composition is in a form chosen from a food supplement, a foodstuff, a drink, a cosmetic product and a medicament.

[0094] In particular, the composition may be in the form of a tablet, capsule, stick, patch, cream, gel, powder, capsule, solution, suspension, emulsion and chewing gum.

[0095] Finally, said composition comprising the combination of the two active ingredients according to any one of the preceding embodiments is intended to be used as a prebiotic composition, preferably the composition is capable of increasing the quantity of bacteria of the genus Akkermansia in the intestinal microbiota of a subject, more preferably of increasing the quantity of bacteria of the species Akkermansia muciniphila in the intestinal microbiota of a subject.

[0096] Thus, the invention also aims at the use of a composition according to any of the preceding embodiments as a prebiotic.

[0097] According to a variant, the invention relates to such a composition for its use as a medicament.

[0098] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples

[0099] Example 1 of composition according to the invention

[0100] A first example of a composition according to the invention is an extract obtained by implementing the method consisting of implementing the following steps: - use of stigmas of Crocus sativus, - grinding using a pin mill, to a size of 250pm, - hydroalcoholic extraction with 60% v / v ethanol, at a rate of 50g of saffron per liter of hydroalcoholic solution, - impregnation on maltodextrin, introduced into the hydroalcoholic solution, - heat treatment in an oven for 48 hours at 40°C.

[0101] The extract obtained is assayed in several molecules with the UHPLC method described in the preamble to the part relating to the examples. The chromatogram obtained is presented in [Fig.l].

[0102] The extract is characterized by: - a safranal concentration of 0.238%, - a Crocine concentration of 3.96%, - a concentration of picrocrocin derivatives of 1.08%, and - a concentration of Flavonoids of 0.25%.

[0103] Example 2 of composition according to the invention

[0104] A second example of a composition according to the invention is an extract obtained by implementing the method consisting of implementing the following steps: - Use of stigmas of Crocus sativus, - grinding using a pin mill, to a size of 250pm, - aqueous extraction, at a rate of 150g of saffron per liter of solution, - solid / liquid separation of the extract obtained - spray drying on maltodextrin support

[0105] The extract obtained is measured in several molecules with the UHPLC method described in the preamble to the part relating to the examples.

[0106] The extract is characterized by: - a safranal concentration of 0.032%, - a Crocine concentration of 3.87%, - a concentration of picrocrocin derivatives of 3.01%, and - a concentration of Flavonoids of 0.27%.

[0107] Example 3 of composition according to the invention

[0108] A third example of a composition according to the invention comprises 30 mg of a saffron extract according to example 2 and 500 mg of a mixture of Akkermansia muci niphila bacteria. Example 3 is obtained by mixing the constituents and then packaged in 2 capsules under conventional conditions of those skilled in the art to reach 2 x 109 CFU per capsule.

[0109] Example 4 of composition according to the invention

[0110] A fourth example of a composition according to the invention comprises 30 mg of saffron extract according to example 1 and 1970 mg of a mixture of Akkermansia muci ni phila, pasteurized at 5 x 108 cells / g of the mixture of Akkermansia muci ni phila, as described for example in patent EP 3 347 030. The composition according to example 4 is obtained by mixing the constituents and then packaged in sachets under conventional conditions of those skilled in the art. [YES] Example 5 of composition according to the invention

[0112] A fifth example of composition according to the invention comprises 2 grams of the composition according to example 4 and 2 g of chicory inulin.

[0113] Example 6 - Evaluation of the effectiveness of the prebiotic composition according to the invention on the microbiota

[0114] The objective of this test is to measure the impact of supplementation with the composition according to example 1, on the microbiota in animals and more particularly on the population of bacteria belonging to the genus Akkermansia.

[0115] To do this, 3 groups of 6 C57 / B16 mice received injections of saline (SHAM) or lipopolysaccharide (LPS) (0.5mg / kg of body weight) every week for 8 weeks, then were fed either with a conventional diet (SHAM and LPS group) or with a diet enriched with the composition according to example 1, at a rate of 44mg / kg of diet (LPS-SAF group).

[0116] At the end of the 8 weeks of treatment, fecal samples were collected for microbiota analysis. Genomic DNA was then extracted from all samples using a commercially available extraction kit. The quality of genomic DNA in all samples was verified by agarose gel electrophoresis and the DNA concentration was measured using a benchtop fluorometer. The V3-V4 hypervariable regions of bacterial 16s RNAs were amplified and sequenced. Sequence analysis was performed using Uparse software. Sequences with >97% similarity were assigned to the same Operational Taxonomy Unit (OTU). Mothur software was used to compare each representative sequence to the SSUrRNA database, itself adapted from the SILVA data. OTU abundance was normalized using a standard sequence corresponding to the sample with the fewest sequences. Alpha (measure of the intrinsic bacterial diversity of each sample) and beta (measure of the diversity of bacterial species between samples) diversity of the microbiota were then assessed using standard measures (using the de Chaol and Shannon H index for alpha diversity and principal coordinate analysis using the Bray-Curtis dissimilarity index for beta diversity). Statistical significance was determined by Kruskal-Wallis or permutational multivariate analysis of variance (PERMANOVA). The LEfSe (Linear discriminant analysis Effect Size) method was also used to determine the characteristics most likely to explain the differences between groups.Finally, gender-level comparisons were performed using classical univariate analysis by Kruskal-Wallis combined with a Dunn's test or a false discovery rate approach to correct for multiple testing.

[0117] The results presented in [Fig. 1] show that alpha diversity is not modified while beta diversity of the microbiota is significantly different (p<0.04) between the groups.

[0118] Example 7 - Analysis of the microbiota by the LEfSe method

[0119] The material and method used is identical to that described in Example 6.

[0120] The results are presented in [Fig.2]. Analysis of the microbiota using the LEfSe method showed that one of the characteristics most likely to explain the differences between the groups was the population of the genus Akkermansia. Indeed, the inventors were able to observe that it is in the LPS-SAF group that bacteria belonging to the genus Akkermansia are statistically the most represented compared to the other two groups.

[0121] Thus, the composition according to the invention is capable of increasing the diversity of the microbiota, in particular of increasing bacteria of the genus Akkermansia.

[0122] Example 8 - Analysis of the abundance of bacteria belonging to the genus Akkermansia

[0123] The material and method used is identical to that described in Example 6.

[0124] The results are presented in [Fig.3]. The univariate analysis on the abundance of bacteria belonging to the genus Akkermansia also revealed that there was a significant difference between the groups (p=0.0061). More specifically, the inventors surprisingly observed a significant difference (p=0.0089) between the group treated with LPS and the group treated with LPS but having received a diet enriched with Crocus Sativus extract. Indeed, when the animals are fed with a diet enriched with Crocus Sativus extract, the abundance of bacteria belonging to the genus Akkermansia reaches a median of 1871 (LPS-SAF group) while it is only 95.50 in the group treated with LPS (LPS group) but having received a conventional diet. This result demonstrates that the use of the composition according to the invention induces an increase in the microbiota of the population of bacteria belonging to the genus Akkermansia. In addition, the statistical analysis showed that there was no significant difference in the abundance of bacteria belonging to the genus Akkermansia between the SHAM group and the LPS-SAF group (518 vs 1871) whereas - although not significant - it seems that the LPS treatment induces a strong reduction in the abundance of this bacteria in the microbiota compared to the SHAM control group (95.50 vs 518). Also, a diet enriched with Crocus sativus extract also makes it possible to maintain the population of bacteria belonging to the genus Akkermansia in an inflammatory condition by increasing their quantity.

Claims

Claims

1. Use of a Crocus sativus extract comprising at least 0.01% safranal, by weight relative to the total weight of the dry matter of the extract, measured by HPLC, as a prebiotic.

2. Use according to the preceding claim, as a prebiotic capable of increasing the quantity of bacteria of the genus Akkermansia in the intestinal microbiota of a subject.

3. Use according to the preceding claim, characterized in that the subject presents or is likely to present an inflammatory profile.

4. Use according to one of claims 2 or 3, characterized in that the quantity of bacteria of the genus Akkermansia is increased by at least 20%, compared to the quantity of bacteria initially present in the intestinal microbiota of the subject.

5. Use according to one of claims 2 to 4, characterized in that the quantity of bacteria of the genus Akkermansia is increased by at least one logarithmic unit within the intestinal microbiota of the subject.

6. Use according to one of the preceding claims, characterized in that the Crocus sativus extract is obtained from the stigmas and / or petals of Crocus sativus.

7. Use according to one of the preceding claims, characterized in that the Crocus sativus extract comprises: - at least 0.01% safranal, by weight relative to the total weight of the dry matter of the extract, measured by HPLC, and / or - at least 1% crocins by weight relative to the total weight of the dry matter of the extract, measured by HPLC method, and / or - at least 500 ppm of flavonoids derived from kaempferol by weight of dry matter of the extract, measured by HPLC method, and / or - at least 0.5% picrocrocin by weight of dry matter of the extract, measured by HPLC method, and / or - a bulking agent chosen from maltodextrin, sugar, silica and gum arabic, intended to encapsulate said Crocus sativus extract.

8. Use according to one of the preceding claims, characterized in that the Crocus sativus extract is obtainable by a process comprising the following steps: - grinding of the Crocus sativus containing safranal, - aqueous or hydroalcoholic extraction or with an organic solvent, - drying of the extract on a bulking agent or mixing with a bulking agent, or - impregnation and encapsulation in the bulking agent in the extraction solution, and drying, and / or heat treatment.

9. Composition comprising at least one extract of Crocus sativus (i) and at least one bacterium belonging to the species Akkermansia muciniphila (ii).

10. Composition according to the preceding claim, characterized in that the bacteria is in pasteurized form.

11. Composition according to one of claims 9 or 10, characterized in that it also comprises at least one additional prebiotic ingredient and / or at least one probiotic bacterium and / or yeast.

12. Composition according to the preceding claim, characterized in that: - the prebiotic ingredient is chosen from rinulin, FOS, GOS, XOS or MOS, and / or - the probiotic bacteria and / or yeast is chosen from bacteria and / or yeast of the genus Lactobacillus, Bifidobacterium, Bacillus, Streptococcus, Saccharomyces, Enterococcus, Pediococcus and Escherichia.

13. Composition according to one of claims 9 to 12, characterized in that the composition is in a form chosen from a food supplement, a foodstuff, a drink, a cosmetic product.

14. Composition according to one of claims 9 to 13, characterized in that the composition is in the form of a tablet, capsule, stick, patch, cream, gel, powder, capsule, solution, suspension, emulsion or chewing gum.

15. Use of a composition according to one of claims 9 to 14, as a prebiotic.

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