Olive oil margins

Abstract

Oleacein Extraction Process. The present invention relates to a process for treating co-products from olive oil production. The present invention also relates to a process for extracting active ingredients of cosmetic, therapeutic, and / or food interest. No Abstract Figure

Description

Title of the invention: Olive oil margins technical field

[0001] The present invention relates to a process for treating co-products from the production of olive oil.

[0002] The present invention also relates to a method for extracting active ingredients of cosmetic, therapeutic and / or food interest. Previous technique

[0003] Over the past few decades, the European and global olive oil sector has been transformed. In 2016, global olive oil production amounted to 3 million tonnes. France, a small producer, distinguishes itself by another factor besides production volume: quality, high-end products, and numerous identified varieties.

[0004] Generally speaking, olive oil extraction processes are characterized by three main steps, including a crushing step, a kneading step, and then a separation step. This last step can be carried out using several methods.

[0005] Thus, four main methods of olive oil extraction are currently used in the world (traditional method, 2-phase, 2-phase / 2 and 3-phase).

[0006] The traditional discontinuous process known as pressing is the oldest but has not been able to meet the growing consumer demand for olive oil.

[0007] Thus, in the 1970s, a continuous extraction process by centrifugal decantation, known as the "three-phase" process, emerged, which uses a large quantity of water to extract olive oil. However, the increase in olive oil production yield generates a very large volume of co-products, including a highly polluting aqueous effluent, olive mill wastewater, or vegetation water.

[0008] The olive mill wastewater from pressing extraction, known as "traditional", remains a by-product with a very high polluting load, but due to the small addition of water during the oil extraction, the volumes of olive mill wastewater produced remain relatively limited compared to other processes such as the three-phase method which generates quantities of olive mill wastewater up to 1200 L for one tonne of crushed olive.

[0009] The extraction method using so-called "water saving" or two-phase decanters requires less water to be added, which limits the volumes of olive mill wastewater discharged (550 to 750 L for one tonne of olives).

[0010] To limit the emission of this effluent, a new system has been implemented: the continuous two-phase extraction system, which consumes significantly less water. The two-phase extraction system is very similar to the three-phase or two-and-a-half-phase extraction system, but does not require the addition of water before or during the mixing stage. Unlike three-phase or two-and-a-half-phase processes, the pomace produced by the two-phase method is moist. However, even moist pomace is considered similar to olive mill wastewater (OMW), as it has a high pollutant load close to that observed with other extraction methods, according to the France Olive report published in 2011.

[0011] Also, even when heavily diluted by the large quantities of water added at the settling tank, and regardless of the extraction method used, this type of olive mill wastewater remains a by-product with a high polluting load.

[0012] Waste reprocessing solutions (composting, spreading, destruction, etc.) have been put in place by the olive oil sector, but the latest bulletin (Guide to good practices for the disposal of by-products October 2011) from France Olive (formerly called AFIDOL) (French Interprofessional Olive Association) reports through its audit the very large volumes and polluting loads specific to olive mill wastewater.

[0013] The composition of olive oil and olive mill wastewater can vary depending on the olive variety, fruit maturity, climatic conditions, storage time, and the extraction method used. The majority of polyphenols are found in the olive mill wastewater (40%), and only 0.3 to 1.5% of the available phenols were found in the olive oil, depending on the system used.

[0014] The predominant phenolic compound in olive mill wastewater is hydroxytyrosol, a compound containing a single phenol group. Other simple phenolic compounds are frequently found in olive oil and in olive mill wastewater, but at lower concentrations; notably tyrosol, elenolic acid, oleuropein, protocatechuic acid, veratric acid, vanillic acid, gallic acid, 4-hydroxyacetic acid, cinnamic acid, paracoumaric acid and syringic acid.

[0015] These molecules are phenolic acid or alcohol compounds possessing one or more phenolic functional groups. They are derived by hydroxylation of benzoic acid (C6-C1) or cinnamic acid (C6-C3) (phenylpropanoid family). Other, more complex phenolic compounds are also found, such as secoiridoids, like oleuropein, which is an ester of elenolic acid and hydroxytyrosol, or molecules having a basic structure similar to that of glycosylated coumaric acid.

[0016] WO2005 / 123603 reports a process for extracting chemical compounds from co-products from olive oil production.

[0017] There remains a need for the treatment of toxic effluents from olive oil production; in particular for the treatment of olive mill wastewater from 2-phase and 3-phase olive oil extraction processes.

[0018] WO2010 / 094860 reports a process for extracting phenolic compounds from low molecular weight from olive or grape vegetation waters.

[0019] There remains a need for new environmentally acceptable olive oil production processes adapted to market needs.

[0020] In particular, there remains a need for industrially applicable production processes.

[0021] There remains a need for valorization of co-products from olive oil production.

[0022] There therefore also remains a need for the treatment of co-products from the production of olive oil; and in particular of olive mill wastewater.

[0023] In particular, there remains a need for detoxification of olive oil margins, the toxicity of which is attributed to their phenolic constituents.

[0024] There also remains a need to identify the pharmaceutical, cosmetic and food properties of olive oil, and its co-products.

[0025] There also remains a need for the extraction of compounds of pharmaceutical, cosmetic and food interest.

[0026] The present invention aims to satisfy these needs. Summary of the invention

[0027] The inventors identified the presence of oleacein (3,4 DHPEA-EDA), a phenolic compound of the secoiridoid family, in olive oil margins.

[0028] To the inventors' knowledge, the presence of oleacein in such olive mill wastewater had, until now, only been identified in trace amounts, and not industrially valorized.

[0029] The inventors were thus able to evaluate the impact of parameters influencing the oleacein content in these olive mill wastewaters, and a sampling protocol was implemented to minimize variability; both in the raw material and in an extraction process.

[0030] These implemented processes remain applicable on an industrial scale.

[0031] The inventors were finally able to confirm the presence of biological activity in fractions obtained, induced by the presence of oleacein as the main active ingredient.

[0032] This extraction process can therefore relate to both processes for treating olive oil margins, but also to processes for extracting oleacein, as well as the valorization of the fractions obtained, particularly in the pharmaceutical, cosmetic and food sectors.

[0033] According to one of its main objects, the present invention relates to a process for treating olive oil wastewater, comprising the following steps: a. supply of an olive oil margin composition, the margin being characterized by a dry matter content of between 3% and 20% of the total mass of the composition; b. adjusting the pH of the composition to an acidic pH, between 3.5 and 5.5; c. enzymatic treatment of the adjusted composition; d. treatment of the composition by microfiltration (MF), and collection of the microfiltered fraction, in particular the microfiltered permeate; e. treatment of the microfiltered fraction by nanofiltration (NF), and collection of the nanofiltered fraction, in particular of the nanofiltered retentate; f. Optionally, treatment of the nanofiltered fraction by centrifugal partition chromatography (CPC) and collection of the purified fraction.

[0034] According to one of its other main objects, the present invention relates to a process for extracting oleacein, comprising the implementation of a process for treating olive oil margins, as defined above, and the presence of at least one step of collecting a fraction comprising oleacein.

[0035] According to one of its other main objects, the present invention relates to an oleacein composition obtained by an extraction process, as defined above, characterized in that it comprises said olive oil margin fraction.

[0036] According to one of its other principal objects, the present invention relates to an oleacein composition as defined above, characterized in that it comprises said fraction of olive oil margin; for its use as a medicinal product.

[0037] According to one of its other main objects, the present invention relates to a cosmetic or food use of an oleacein composition as defined above. Brief description of the drawings

[0038] [Fig. 1] represents a pre-treatment protocol for olive oil wastewater up to the microfiltration stage. The stages of collecting the olive mill wastewater, at the outlet of the decanter, then adjusting the collected olive mill wastewater to an acidic pH.

[0039] [Fig.2] represents a comparison of the viscosity with and without treatment enzymatic (300 ppm, 30 °C, 1 h), the treated fractions including four enzymatic preparations with pectinolytic activity of defatted olive mill wastewater from a varietal blend of olives after three-phase extraction. The y-axis shows the corresponding viscosity, expressed in mPa*s (at 100 s⁻¹). The tested olive mill wastewaters correspond, From left to right, at: (i) a milling wastewater without enzymatic treatment, (ii) a milling wastewater enzymatic with Pectinex Ultra SPL, (iii) a milling wastewater enzymatic with Pectinex Ultra Pulp, (iv) a milling wastewater enzymatic with Ultrazyme AFP, (v) a milling wastewater enzymatic with Rhodopect 100 PTE. Detailed description

[0040] The term "oleacein" herein specifically refers to the phenolic compound of the secoiridoid family, 3,4-DHPEA-EDA, whose CAS number is 149183-75-5, with the following structural formula:

[0041] [Chem.l]

[0042] This term is likely to encompass all oleacein salts, and in particular all pharmaceutically acceptable oleacein salts.

[0043] Unless otherwise indicated, this term does not include oleacein analogues found, in particular, in olive mill wastewater or olive oils, such as oleuropein and oleuropein aglycone, which are likely to be at the origin of the formation of oleacein during the pressing of olives.

[0044] Document WO2017122034 teaches processes for increasing the levels of certain polyphenols, including oleacein, in olive oil.

[0045] Document WO2014012871 teaches biological properties, including pharmaceutical properties, of oleacein as an active ingredient.

[0046] As detailed in the examples below, the inventors demonstrate that olive oil mill wastewater, normally considered as toxic effluents, is likely to contain significant amounts of oleacein.

[0047] Also surprisingly, the inventors highlight a process for treating these olive oil margins, the final products of which are characterized by high oleacein content, compatible with valorization in the pharmaceutical, cosmetic and food fields.

[0048] This treatment process thus makes it possible to obtain compositions enriched in oleacein, with high biological potential, stable and exhibiting good reproducibility.

[0049] This point is all the more surprising since several analogues of oleacein, and degradation products, have already been reported in olive mill wastewater or olive oil.

[0050] In this regard, and to the inventors' knowledge, oleacein has so far only been characterized in olive oil margins in small quantities.

[0051] The degradation of oleacein can, in particular, produce two by-products: EDA and hydroxytyrosol. Finally, oleocanthal, found in olive oil, is the closest structural analogue of oleacein.

[0052] Thus, document WO2017122034 teaches processes for increasing the levels of certain polyphenols, including oleacein, in olive oil.

[0053] Document WO2014012871 teaches biological properties, including pharmaceutical properties, of oleacein as an active ingredient.

[0054] The inventors therefore propose a process for treating these olive mill wastewaters, with a view to obtaining a product with high biological potential; where appropriate, this treatment process can therefore be associated with a process for extracting and / or preparing oleacein, as an active ingredient.

[0055] This process for treating olive oil wastewater is characterized, at least, by: i. the supply of an olive oil wastewater composition characterized by a dry matter content of between 3% and 20% of the total mass of the composition; and ii. the implementation of an enzymatic treatment step, a microfiltration (MF) step, and a nanofiltration (NF) step.

[0056] Thus, according to one of its main objects, the present invention relates to a process for treating olive oil wastewater, comprising the following steps: a. supply of an olive oil margin composition, the margin being characterized by a dry matter content of between 3% and 20% of the total mass of the composition; b. adjusting the pH of the composition to an acidic pH, between 3.5 and 5.5; c. enzymatic treatment of the adjusted composition; d. treatment of the composition by microfiltration (MF), and collection of the microfiltered fraction; e. treatment of the microfiltered fraction by nanofiltration (NF), and collection of the nanofiltered fraction.

[0057] Depending on the raw material and the characteristics of the collected fractions, this treatment process may also include other steps.

[0058] Advantageously, said process is likely to include a step of treatment by centrifugal partition chromatography (CPC) and collection of the purified fraction.

[0059] According to this particular embodiment, the present invention relates to a process for treating olive oil wastewater, comprising the following steps: a. supply of an olive oil margin composition, the margin being characterized by a dry matter content of between 3% and 20% of the total mass of the composition; b. adjusting the pH of the composition to an acidic pH, between 3.5 and 5.5; c. enzymatic treatment of the adjusted composition; d. treatment of the composition by microfiltration (MF), and collection of the microfiltered fraction; e. treatment of the microfiltered fraction by nanofiltration (NF), and collection of the nanofiltered fraction; f. treatment of the nanofiltered fraction by centrifugal partition chromatography (CPC) and collection of the purified fraction.

[0060] According to certain embodiments of the process for treating olive oil wastewater, the said process further comprises one or more sieving steps; in particular one or more sieving steps before the microfiltration treatment step.

[0061] According to certain embodiments of the process for treating olive oil pomace, said process includes one or more sieving steps after the enzymatic treatment step, and before the microfiltration treatment step.

[0062] According to certain embodiments of the process for treating olive oil wastewater, said process consists of steps a), b), c), d) and e).

[0063] According to certain embodiments of the process for treating olive oil wastewater, said process consists of steps a), b), c), d), e) and f).

[0064] According to certain embodiments of the process for treating olive oil wastewater, step b) immediately follows step a), without an intermediate step.

[0065] According to certain embodiments of the process for treating olive oil pomace, step c) immediately follows step b), without an intermediate step.

[0066] According to certain embodiments of the process for treating olive oil wastewater, step d) immediately follows step c), without an intermediate step.

[0067] According to certain embodiments of the process for treating olive oil wastewater, step e) immediately follows step d), without an intermediate step.

[0068] According to certain embodiments of the process for treating olive oil pomace, step f) immediately follows step e), without an intermediate step.

[0069] Said microfiltered fraction, said nanofiltered fraction, and where applicable said purified fraction may be characterized by the presence of oleacein.

[0070] The fractions characterized by the presence of oleacein are chosen from: microfiltered permeate, nanofiltered permeate, microfiltered retentate and nanofiltered retentate.

[0071] The fractions characterized by the presence of oleacein are, in particular, the microfiltered permeate and the nanofiltered retentate from said treatment and extraction processes.

[0072] An olive oil margin composition, characterized by a dry matter content of between 3% and 20% of the total mass of said composition, is likely to be characterized by a dry matter content of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% of the total mass of said composition.

[0073] According to certain particular embodiments of the treatment process according to the invention, said olive mill composition is capable of being characterized by a dry matter content equal to or greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19% of the total mass of said composition.

[0074] According to certain particular embodiments of the treatment process according to the invention, said olive mill composition is capable of being characterized by a dry matter content equal to or less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% of the total mass of said composition.

[0075] According to certain particular embodiments of the treatment process according to the invention, said composition of olive oil margins is characterized by a dry mass content of between 4% and 13% of the total mass of the composition.

[0076] According to certain particular embodiments of the treatment process according to the invention, said olive mill wastewater is derived from an olive oil production process in a two-phase, two-phase or three-phase system; in particular derived from a two-phase or three-phase system process.

[0077] According to certain particular embodiments of the treatment process according to the invention, said olive millings are derived from a variety selected from: Aglandau, Bouteillan, Cailletier, Cayetroux, Cayon du Var, Clermontaise, Ghjermana, Grossane, Olivière, Lucques, Négrette, Petit Ribier, Picholine, Rougette de l'Ardèche, Sabine, Salonenque, Tanche, Verdale de l'Hérault, Zinzala; in particular selected from: Bouteillan, Négrette, Aglandau, Picholine, Lucques and Olivière; or from a varietal mixture, such as a mixture comprising any one of said varieties.

[0078] According to certain preferred embodiments of the treatment process according to the invention, said olive mill wastewater comes from the Picholine variety, or from a varietal mixture including the Picholine variety.

[0079] The step of adjusting the pH of the composition to an acidic pH, included in the treatment process according to the invention, may include an adjustment to any acidic pH between 3.5 and 5.5; this includes all pH values ​​between these two ranges, namely: 3.5; 3.6; 3.7; 3.8; 3.9; 4.0; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; 5.0; 5.1; 5.2; 5.3; 5.4; 5.5.

[0080] According to certain particular embodiments of the treatment process according to the invention, the pH of the composition is adjusted between 4.0 and 4.5.

[0081] According to certain embodiments of the treatment process according to the invention, the enzymatic treatment step, in particular by maceration, is carried out in the presence of one or more pectinolytic enzymes, or pectinases.

[0082] Examples of pectinolytic enzymes, or enzyme mixtures according to the invention, are known to those skilled in the art. Such enzyme mixtures may advantageously comprise one or more pectinolytic enzymes, chosen from polygalacturonases, pectin lyases, pectin methyl esterases, or any combination thereof.

[0083] In particular, examples of commercial compositions that can be implemented in a process of the invention can be chosen from: Pectinex® Ultra SP-L (Novozymes), Pectinex® Ultra Pulp (Novozymes), Pectinex® Ultra AFP, Rohapect® MC (AB Enzymes).

[0084] According to an exemplified embodiment, an enzymatic composition that can be implemented in a process of the invention can consist of, or comprise, a Rohapect® PTE100 composition (AB Enzymes).

[0085] According to certain embodiments of the treatment process according to the invention, the enzymatic treatment step being carried out at a temperature equal to or greater than 30°C, for example between 30°C and 55°C; which is therefore likely to include 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55°C.

[0086] According to certain particular embodiments of the treatment process according to the invention, the enzymatic treatment step is carried out at a temperature equal to or greater than 45°C.

[0087] According to certain embodiments of said process, the microfiltration (MF) step can be carried out through a ceramic membrane.

[0088] According to certain particular embodiments of said process, the microfiltration (MF) step can be carried out through a ceramic membrane selected from: - a membrane characterized by an active layer of type A12O3; - a membrane characterized by an active layer of the TiO2 type; - a membrane characterized by an active layer of the ZrO2 type. - a membrane characterized by an active layer of the SiC type.

[0089] According to certain particular embodiments of said process, the microfiltration (MF) step is carried out through a membrane having an average pore diameter of between 0.1 and 2 pm; in particular between 0.1 and 1.4 pm.

[0090] According to certain embodiments of the treatment process according to the invention, the microfiltration (MF) step being carried out through a ceramic membrane; with an average pore diameter of between 0.1 and 1.4 pm; the microfiltration (MF) step being in particular a tangential microfiltration (MFT) step.

[0091] According to certain embodiments of the treatment process according to the invention, said process does not include a step of separating the solid phase and the liquid phase before the microfiltration step.

[0092] According to certain embodiments of the treatment process according to the invention, the nanofiltration step is carried out through a membrane having a cutoff threshold less than 1000 Da, in particular between 100 and 1000 Da; for example less than 500 Da, in particular between 100 and 500 Da.

[0093] According to certain particular embodiments of the treatment process according to the invention, the nanofiltration step is carried out through a membrane having a cut-off threshold between 100 Da and 500 Da, for example between 200 and 500 Da.

[0094] Examples of nanofiltration membranes may be chosen from the following group: NF270 (Dow Filmtec), NF200 (Dow Filmtec), NF90 (Dow Filmtec), TS80 (TriSep), TS40 (TriSep), XN45 (TriSep), UTC20 (Toray), TR60 (Toray), DK (SUEZ ((GE)), DL (SUEZ ((GE)), GE (SUEZ ((GE)), HL (SUEZ ((GE)), NFX (Synder), NFW (Synder), NFG (Synder), TFC SR100 (Koch), SR3D (Koch), SPIRAPRO (Koch) ESNA1 (Nitto-Denko), NTR7450 (Nitto-Denko).

[0095] Examples of nanofiltration membranes can be chosen from polyamide, polypiperazineamide, polysulfone, polyethersulfone, and composite membranes thereof.

[0096] According to certain embodiments of the treatment process according to the invention, said compound comprises at least one step of collecting a fraction comprising oleacein; in particular at least one step of collecting a fraction comprising oleacein from the nanofiltered fraction.

[0097] The invention also relates, according to one of its main objects, to a process for extracting oleacein, comprising the implementation of a process for treating olive oil margins according to any one of the preceding claims, and the presence of at least one step of collecting a fraction comprising oleacein.

[0098] According to certain embodiments of the extraction process according to the invention, said extraction process is characterized in that it comprises at least one step of collecting a fraction comprising oleacein from the nanofiltered fraction.

[0099] According to certain particular embodiments, said treatment and extraction processes may also include one or more additional steps. In particular, said processes may also include one or more additional steps selected from: an additional microfiltration step, an additional nanofiltration step, an ultrafiltration step, a reverse osmosis step.

[0100] The invention also relates, according to one of its main objects, to an oleacein composition obtained by said extraction process. Applications of oleacein, as an active ingredient

[0101] According to one embodiment, the invention relates to the implementation of oleacein, as an active ingredient, for its use as a medicinal product.

[0102] According to an alternative embodiment, the invention relates to a method of preparing a drug, and / or a pharmaceutical composition comprising the use of oleacein as an active ingredient.

[0103] Most advantageously, the oleacein used as an active ingredient to comprise, or even be made up of, a fraction derived from olive oil margins, and particularly a fraction derived from olive oil margins obtained according to a process of the invention.

[0104] According to one embodiment, the invention therefore relates to the implementation of oleacein, as an active ingredient, for the treatment or prevention of a disorder in an individual.

[0105] According to one embodiment, the invention therefore relates to the use of oleacein, as an active ingredient, for the treatment or prevention of a disorder in an individual, said disorder being chosen from: - Disorders associated with heat stress: - disorders associated with oxidative stress, such as endogenous oxidative stress or exogenous.

[0106] The said disorders may be, in particular, disorders of a pathological or non-pathological nature, such as disorders of a cosmetic nature.

[0107] According to one embodiment, the invention relates to the implementation of oleacein, as an active ingredient, for the treatment or prevention of a disorder selected from: an inflammatory disorder, a cardiovascular disorder, a skin disorder, a proliferative disorder, for example cancer, a neurological disorder, diabetes, atherosclerosis, obesity.

[0108] According to one embodiment, the invention relates to the implementation of oleacein, as an active ingredient, for the treatment or prevention of an inflammatory disorder; for example, an immune-mediated inflammatory disorder and / or an inflammatory bowel disorder, selected from: chronic bowel disease, multiple sclerosis, chronic obstructive pulmonary disease, Crohn's disease, ankylosing spondylitis, systemic lupus erythematosus, psoriasis, psoriatic arthritis and myocarditis.

[0109] According to an alternative embodiment, the invention relates to the implementation of oleacein, as an active ingredient, for non-therapeutic use.

[0110] According to one embodiment, the invention relates to a method of preparing a food composition, and / or a cosmetic composition comprising the use of oleacein as an active ingredient.

[0111] According to one embodiment, the invention therefore relates to a cosmetic or food use of an oleacein composition, as an active ingredient, such as a oleacein composition obtained from olive oil margins, and particularly obtained from margins, according to a process of the invention.

[0112] According to one embodiment, the invention therefore relates to the implementation of oleacein, as an active ingredient, for the treatment or prevention of a cosmetic disorder associated with age, such as a skin disorder, chosen from: wrinkles, fine lines, sagging skin, loss of skin density, loss of skin firmness, photo-aging. Definitions

[0113] The terms used in this description are used with their usual meaning in the relevant technical field and in light of the context of the description in which the terms are used. Some terms are discussed further below, or elsewhere in the description, to provide additional guidance regarding the invention and its implementation. The following definitions are provided for the purposes of the description and the claims.

[0114] The description of the various embodiments of the invention includes embodiments including "comprising," "having," "consisting of," and "consisting essentially of." The words "having" and "comprising," or variants such as "has," "have," "includes," or "comprising," should be understood as implying the inclusion of the indicated element(s) (such as an element of a composition or a method step) but not the exclusion of other elements. The term "consisting of" implies the inclusion of the indicated element(s), to the exclusion of any additional element(s). The expression "consisting essentially of" implies the inclusion of the indicated elements, and possibly other elements when the other elements do not materially affect the fundamental and novel features of the invention.Depending on the context, the term "include" can also strictly refer to the stated features, whole numbers, steps, or components, and therefore, in this case, it can be replaced by "consist of".

[0115] The term "approximately" as used here with respect to a numerical value refers to the usual range of error for the value in question, as it is normally identified by a person skilled in the art in the relevant technical field. The use of the term "approximately" with respect to a specific value or parameter includes and describes that value or parameter as such. The term "approximately" refers to ±10% of a given value. However, whenever the value in question refers to an indivisible object that would lose its identity upon subdivision, then "approximately" refers to ±1% of the indivisible object.

[0116] The term "individual" or "patient" as used in this text refers in particular to a mammal. The mammals considered include, in particular, Domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-humans), rabbits, and rodents (e.g., mice and rats). In one particular embodiment, an individual, or patient, is a human being.

[0117] In the context of the present invention, the terms "prevent," "prevention" (and variations thereof) with respect to a physiological disorder or disease refer to the prophylactic treatment of the disease or disorder, for example, in an individual suspected of having that disease or disorder, or at risk of developing that disease or disorder. Prevention includes, but is not limited to, preventing or slowing the development of the disease, and / or maintaining one or more symptoms of the disease at a desired or reduced level. The term "prevent" does not require the complete elimination of the possibility or probability of the disease or disorder occurring. Rather, it refers to reducing the risk or probability of a given phenomenon to a lesser degree.As indicated, prevention can be complete, meaning the absence of symptoms or detectable disease, or partial, such that there are fewer symptoms or the symptoms are of lesser intensity.

[0118] In the context of the present invention, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of the pathological processes under consideration. The specific amount that is therapeutically effective can be readily determined by a physician and may vary depending on factors such as the type and stage of the pathological processes under consideration, the patient's medical history, sex, weight, and age, diet, and the administration of other therapeutic agents.

[0119] For the purposes of the invention, the term "significantly" or any derived terms, used in the context of a change, means that the observed change is notable or has statistical significance.

[0120] In the context of the present invention, the terms “treat,” “treatment,” “therapy,” or “therapeutic” refer to the administration or consumption of an active ingredient for the purpose of curing, relieving, reducing, mitigating, or improving a disease or pathological disorder, or one or more associated symptoms, or to prevent or slow the progression of such symptoms or disease, or to halt the development of such symptoms, or disease, or pathological disorder in a statistically significant manner. More specifically, “treat” or “treatment” includes any approach to achieving a beneficial effect or desired outcome with respect to a disease in an individual. The beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or improvement of the disease or one or more symptoms of such a disease; the decrease or reduction of the extent of the disease, stabilization, that is, the absence of aggravation of a disease, or of one or more symptoms of such a disease; the prevention of a disease, or of one or more symptoms of such a disease; the prevention of the spread of a disease, or of one or more symptoms of such a disease; the slowing of a disease, or of one or more symptoms of such a disease or of the progression of one or more symptoms of such a disease; the reduction of the recurrence of an associated disease, or of one or more symptoms of such a disease; and the interruption of a disease, or of one or more symptoms of such a disease.In other words, "treatment" as used here includes any cure, improvement, reduction, or cessation of a disease, or of one or more symptoms of such a disease. A "reduction" of a symptom or disease means a decrease in the severity or frequency of the disease or symptom, or the elimination of the disease or symptom.

[0121] By "pharmacologically acceptable" or "physiologically acceptable" is meant that the vehicle (carrier, diluent, or excipient) must be compatible with the other ingredients of the formulation and not harmful to the individual to whom the composition containing it is administered. A pharmaceutically acceptable vehicle is one recognized as meeting, in particular, the criteria of safety, compatibility, and inertness required for use in the pharmaceutical field. Examples of pharmaceutically acceptable vehicles include sterile water, saccharides such as sucrose or saccharose, starches, sugar alcohols such as sorbitol, polymers such as PVP or PEG, lubricating agents such as magnesium stearate, preservatives, and coloring or flavoring agents.

[0122] In the context of the present invention, the term "physiologically acceptable vehicle" is intended to designate any substance or composition compatible with the organism of the individual to whom an active ingredient of the invention is to be administered. In particular, a physiologically acceptable vehicle is a substance or composition whose administration to an individual does not result in significant adverse effects. It may be, for example, a non-toxic solvent such as water or an aqueous saline solution. In particular, such a vehicle is compatible with oral or rectal administration, and preferably is suitable for oral administration.

[0123] By "pharmacologically acceptable salt" is meant a salt that retains the desired biological activity of the parent compound and does not impart undesirable toxic effects. Acid addition salts and base addition salts are examples such salts. Acid addition salts include nontoxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, and hydroxyalkanes. Salts derived from nontoxic organic acids such as acids, aromatic acids, and aliphatic and aromatic sulfonic acids are included. Base addition salts include, for example, alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and others. Salts derived from toxic organic amines are included.

[0124] The list of sources, ingredients and components indicated below are understood to be described in such a way that all combinations and mixtures of these are also envisaged within the scope of the present invention.

[0125] It is understood that each maximum numerical limitation given in the description includes each lower numerical limitation, as if such lower numerical limitations were expressly stated. Each minimum numerical limitation given in this description includes any upper numerical limitation, as if such upper numerical limitations were expressly stated herein. Each numerical range given throughout the description includes each narrower numerical range included within such a wider numerical range, as if such narrower numerical ranges were all expressly stated.

[0126] All lists indicated in the description, such as, for example, lists of ingredients, are intended to be and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as elements "selected from the group consisting of" ... list of elements ... "and their combinations and mixtures".

[0127] Reference may be made herein to trade names of components comprising various ingredients used in this description. The inventors do not intend to be limited to materials under any particular trade name. Equivalent materials (for example, those obtained from a different source under a different name or reference number) to those indicated herein by a trade name may be substituted and used in the description below.

[0128] The pharmaceutical compositions described herein can also be implemented in combination therapy, i.e. in combination with other active agents.

[0129] In some cases, the pharmaceutical compositions described herein may include other compounds, agents and / or drugs used to treat conditions such as cancer, autoimmune disease or inflammatory disease. These compounds, agents and / or drugs may include, for example, chemotherapeutic agents, small molecule agents or antibodies that stimulate an immune response against a given cancer.

[0130] The pharmaceutical compositions described herein may comprise one or more pharmaceutically acceptable salts.

[0131] The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, oxidizing agents such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and others; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and others.

[0132] Examples of suitable aqueous or non-aqueous supports that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by preserving the required particle size in the case of a dispersion and by using surfactants.

[0133] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The prevention of microorganisms can be ensured both by the sterilization methods described above and by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and others. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, can delay the absorption of injectable pharmaceutical forms.

[0134] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such carriers and agents for pharmaceutically active substances is well known in the field. Except to the extent that a conventional medium or agent is incompatible with the active ingredient, its use in the pharmaceutical compositions described herein is envisaged. Additional active substances may also be incorporated into the compositions.

[0135] A pharmaceutical composition must generally be sterile and stable under manufacturing and storage conditions. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for a high concentration of the drug. The carrier may be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and others), and suitable mixtures thereof. Appropriate fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition.Delayed absorption of injectable compositions can be caused by the inclusion in the composition of an absorption-delaying agent, for example, monostearate salts and gelatin.

[0136] Sterile injectable solutions can be prepared by including the active compound, in the required quantity, in a suitable solvent, optionally with one or a combination of the ingredients listed above, and then sterilizing by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is a vacuum drying method, in which a powder of the active ingredient plus any other desired ingredient is produced from the previously sterilized filtered solution, followed by lyophilization.

[0137] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form may vary depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. According to some embodiments, the amount of active ingredient is from about 0.01% to about 99% of the amount of the final composition, preferably from about 0.1% to about 70% of the amount of the final composition, most often combined with a pharmaceutically acceptable carrier.

[0138] Dosage regimens are adjusted to obtain the desired optimal response (e.g., a therapeutic response). According to some embodiments, a single bolus administration is possible and several divided doses may be administered. may be administered over a long period of time, or the dose may be reduced or increased proportionally as indicated in a situation of imminent treatment. Preferably, the formulation of parenteral compositions is in unit doses, particularly to facilitate administration and dosage uniformity.

[0139] According to some embodiments, a unit dose means a physically suitable unit as a single dose for the individual to be treated; each unit, combined with the required pharmaceutical support, produces the desired therapeutic effect.

[0140] According to some embodiments, the pharmaceutical composition is used for prophylactic or therapeutic treatment. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over long periods at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until disease progression is slowed or halted, preferably until the patient experiences partial or complete improvement in disease symptoms. Thereafter, the patient may be placed on a prophylactic regimen.

[0141] The actual dosage levels of the active ingredients in the pharmaceutical compositions according to the invention are not toxic to the patient in order to obtain the desired therapeutic response for the individual, in particular the composition and the route of administration. It is possible to vary them to obtain an effective amount of the active ingredient. The selected dosage level depends on the particular composition used, or the activity of its ester, salt, or amide, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with a particular composition, the age, sex, weight, condition, general health, and medical history of the individual being treated, as well as similar factors well known in the medical field.The dosage level may also vary depending on various pharmacokinetic factors.

[0142] According to some embodiments, a therapeutically effective dose can prevent or delay the onset of a disease. For example, laboratory tests used to diagnose a disease include chemistry, hematology, serology, and radiology. Accordingly, clinical or biochemical tests that monitor any of the above can be used to determine whether a particular treatment is a therapeutically effective dose for treating the disease. A person skilled in the art can determine such amounts based on factors such as that the size of the individual, the severity of the individual's symptoms, and the particular composition or route of administration chosen.

[0143] The pharmaceutical compositions according to the invention can be administered by one or more methods known in the field, by one or more routes of administration. As a person skilled in the art will understand, the route and / or method of administration will vary depending on the desired result.

[0144] The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The therapeutic composition can be administered using medical devices known in the field. Examples.

[0145] Example 1: validation of the process of extracting oleacein from olive mill wastewater.

[0146] After evaluating the impact of various parameters that could influence the oleacein content in olive mill wastewater, a sampling protocol was implemented in order to minimize the variability of the compound content in the raw material: olive mill wastewater.

[0147] The objective was to determine the presence and quantity of oleacein in several varietal olive mill wastewaters (Bouteillan, Négrette, Aglandau, Picholine, Lucques and Olivière) and according to several parameters (maturity and extraction method)

[0148] The results showed that the Picholine variety was the one that allowed the greatest quantity of the target compound to be obtained regardless of the extraction method used (2 phases, 2 phases or 3 phases) and regardless of the level of maturity of the olives.

[0149] The main objective being to obtain a purified oleacein extract, several extraction and purification methods were tested, using as a common raw material the olive mill wastewater from this Picholine variety.

[0150] Pre-treatment step: stabilization of olive mill wastewater by acidification.

[0151] This step begins at the settling tanks (the olive mill wastewater is collected directly from the outlet of the centrifuges or settling tanks). The olive mill wastewater characterized by a dry matter content of between 3% and 20% of its total mass is collected.

[0152] The first step common to all pre-treatment methods makes it possible to obtain a purified extract in which the stability of the target active ingredient (Foleacein) is controlled.

[0153] Acidification of olive mill wastewater helps to stabilize the product at microbiological and chemical levels, oleacein being more stable in an acidic environment.

[0154] This stabilization step here requires acidification of the olive mill wastewater to acidic pH (4.3), in particular using sulfuric acid.

[0155] Immediately after the pH adjustment step, a de-oiling step can advantageously be implemented by centrifugation.

[0156] According to a particular embodiment, the use of a centrifugation system allows for the additional extraction of residual oil present in the olive mill wastewater. In the comparative example described here, the wastewater is centrifuged at 34,000 g for 10 minutes at 4 °C, and then the oily supernatant is removed using a vacuum pump. Enzymatic processing stage

[0157] The effectiveness of the enzymatic treatment / liquefaction step is assessed by measuring the viscosity of the olive mill wastewater, a factor directly impacting the performance of the membrane filtration steps. Viscosity measurements at a shear rate of 100 s⁻¹ performed on an Anton Paar MCR 301 rheometer are illustrated in [Fig. 2].

[0158] They are representative of an improvement in the physico-chemical properties of the tested olive mill wastewater after contact with an enzymatic preparation with pectinolytic activities.

[0159] These results highlight a significant decrease in viscosity (by a factor of 2.7 to 2.1 depending on the type of pectinases used) after enzymatic liquefaction of olive mill wastewater obtained from a three-phase extraction of olives from a varietal blend. The Pectinex Ultra SPL and Rohapect 100PTE preparations, which have different compositions of pectinolytic enzymes, are particularly effective. They allow for a significant decrease in viscosity (2.7 to 2.2), associated with a substantial increase in the density of the filtration stream. Microfiltration stage

[0160] To evaluate the operating conditions of the tangential microfiltration step enabling the best filtration flux densities to be obtained, tests without concentration, i.e. with a mass reduction factor of 1, were carried out in a laboratory installation having a nominal volume of 3 L.

[0161] Several membrane-transmembrane pressure pairs were selected. Four tubular membranes were tested at transmembrane pressures varying between 1 and 4 bar with a tangential velocity close to 5 m / s and a temperature of 30 °C.

[0162] The membranes tested, in particular of the ceramic type, include: - an active layer of type A12O3, characterized by a pore diameter of 0.2 pm and a membrane surface area of ​​55 cm2; - an active layer of the TiO2 type, characterized by a pore diameter of 0.2 pm and a membrane surface area of ​​55 cm2; - an active layer of the ZrO2 type, characterized by a pore diameter of 0.2 pm and a membrane surface area of ​​55 cm2; - SiC type active layer, characterized by a pore diameter of 0.6 pm and a membrane surface area of ​​42 cm2.

[0163] In order to confirm the results obtained, the performance of the microfiltration step was evaluated according to the type of pretreatment of the olive mill wastewater (2 phases k? or 3 phases).

[0164] These results confirm the interest of the enzymatic treatment which makes it possible to increase the permeate flux density; in particular with TiO2 type membranes, because the flux densities obtained remain high, even at low pressures (2 bar) which is of interest at the energy level.

[0165] With this membrane, the permeate flux density after enzymatic maceration is between 90 kg⁻¹h⁻²•m² without oil removal and 130 kg⁻¹h⁻²•m² with oil removal, both values ​​compatible with an industrial application.

[0166] Given these good performances, it is therefore possible to choose only the enzymatic pretreatment in order to limit the number of preliminary steps to filtration and thus to limit the cost and complexity of the process (preliminary steps applicable directly in mill).

[0167] Indeed, according to one embodiment, it is advantageous to do away with the step of removing the residual oil which leads to mass losses (estimated at 10-15%), and can promote the degradation of the target compound.

[0168] Not de-oiling / centrifuging the olive mill wastewater prior to the enzymatic treatment and / or microfiltration step also allows for significant time savings, improved yield and economic gains on the proposed process.

[0169] Under optimized operating conditions, the microfiltration step does not result in any loss of oleacein, with its retention being less than 8%. Dry matter and other polyphenols are retained to a greater extent, allowing for purification of the target compound. Finally, the clarification of the enzymatically treated crude olive mill wastewater is complete.

[0170] The influence of the type of collected olive mill wastewater (2 phases / 2 and 3 phases) on microfiltration performance was also studied, given that the characteristics of the initial raw olive mill wastewater can vary depending on the variety of olives, their stage of maturity or the oil extraction process carried out at the mill.

[0171] The study showed that the total dry matter (DM) content appears to increase slightly with olive maturity and differs depending on the mill extraction process. The dry matter of the olive mill wastewater tested in this study is generally between 4 and 9.3%, in particular between 4 and 7%, in olive mill wastewater from a two-phase U2 extraction and from 7.9 to 12.4% with a three-phase extraction.

[0172] Furthermore, the concentration of oleacein is about half as high in olive mill wastewater from a two-phase U2 extraction (0.2 to 1.2 g / kg) as with a three-phase extraction (1.8 to 2.6 g / kg).

[0173] Knowing that a variation in dry matter content can significantly impact the tangential microfiltration step, a comparative study of microfiltration performance was carried out, and is reproduced below in Table 1.

[0174] [Tables] Extraction type: 3-phase, two-phase Yi Crude olive mill wastewater dry matter (%) 8.9 3.0 Oleacein (g / kg crude olive mill wastewater) 1.9 0.2 Standard average permeate flux density Jp (kg·h⁻²·m²) 88 170 Dry matter retention (%) 25 30 Oleacein retention (%) 2 0 Oleacein stability (%) 100 82

[0175] The microfiltration / extraction step is carried out using a TiO2 membrane (TAMI) at 30 °C, without concentration and after pretreatment of raw olive mill wastewater with 300 ppm of Pectinex Ultra SPL mixture for 30 min at 30 °C. The olive mill wastewaters tested are all from the Picholine variety.

[0176] Optimization of the coupling of enzymatic treatment / tangential microfiltration

[0177] The temperature used for the enzymatic pretreatment, upstream of the microfiltration step, has already been optimized. A study of the influence of temperature on microfiltration performance was carried out.

[0178] The results in Table 2 confirm that increasing the filtration temperature from 30 to 50 °C improves performance by increasing the flow density without affecting the stability of the target compound. The results of the comparative study are shown in Table 2.

[0179] [Tables2] Temperature (°C) Average Jp (kg.h'.m2) Dry matter retention (%) Oleacein retention (%) Oleacein stability 1 to 2h (%) Total polyphenol retention (%) 30 129 19 6 98 22 50 164 27 0 100 35

[0180] The microfiltration step is carried out as indicated in Table 1. The total polyphenol retention value is evaluated by the Folin-Ciocalteu method assay.

[0181] The increase in average flux density is explained by a decrease in the viscosity of the olive mill wastewater at 50 °C. The retention of oleacein is not affected by the process temperature, unlike the retention of dry matter and total polyphenols, which is slightly increased, thus favoring the purification of the target compound.

[0182] These results therefore highlight the advantageous properties of coupling enzymatic treatment / tangential microfiltration on a preparation of olive mill wastewater comprising a percentage of dry matter in accordance with the invention.

[0183] The proposed microfiltration process is sufficiently robust to allow the treatment of different types of olive mill wastewater (in particular 3-phase and 2-phase Yï). Thus, for all types of olive mill wastewater, the cumulative flux densities are greater than 50 kg·h⁻² and are therefore compatible with industrial application. Furthermore, the results confirm that olive mill wastewater with a lower total dry matter content (2-phase Yi extraction) is more favorable in terms of flux.

[0184] Analysis of the olive mill wastewater before and after microfiltration also ensures that there is little or no retention or degradation of the target compound during each of the tests. The characteristics of this olive mill wastewater, obtained from concentration microfiltration tests under optimized conditions (means and standard deviations over 3 replicates), are shown in Tables 3 and 4 below, respectively using raw or clarified olive mill wastewater after microfiltration.

[0185] [Tables3] Origin of the margins: Initial crude margins DM (%) SIS (%) PPT (g / kg DM) Oleacein concentration (g / kg eMF) Average purity relative to DM (g / kg eMS) Average purity relative to PPT (g / 100g PPT) 2-phase extraction: 6.2 (0.1) 1.0 (0.1) 6.9 (0.2) 1.1 (0.1) 18.3 (0.6) 16.4 (1.0) 2-phase extraction: 7.7 (0.1) 0.9 (0.1) 7.9 (0.1) 0.8 (0.1) 10 (0.2) 9.7 (0.3) 3-phase extraction: 9.2 (0.1) 1.1 (0.1) 11.4 (0.1) 1.1 (0.1) 11.6 (1.3) 9.4 (1.1) 3-phase extraction 12.4 (0.1) 2.0 (0.1) 12.6 (0.5) 3.3 (0.1) 26.7 (1.2) 26.3 (2.1)

[0186] [Tables4] Origin of clarified margins after microfiltration MS (%) PPT (g / kg of MF) Oleacein concentration (g / kg of MF) Average purity MS (g / kg of MF) Average purity PPT (g / 100g PPT) 2-phase extraction ^2 4.6 (0.1) 5.0 (0.2) 0.9 (0.1) 19.9 (0.4) 18.4 (1.0) 2-phase extraction ^2 6.0 (0.1) 6.4 (0.4) 0.9 (0.1) 15.0 (0.7) 14.0 (1.3) 3-phase extraction 7.7 (0.1) 9.7 (0.1) 1.3 (0.2) 16.2 (2.2) 12.9 (1.9) 3-phase extraction 10.1 (0.1) 9.6 (0.2) 3.8 (0.1) 37.3 (0.3) 39.4 (1.2)

[0187] MS: total dry matter; SIS: insoluble solids in suspension; PPT: total polyphenols; MF: fresh matter.

[0188] With the data obtained from the 4 concentration trials carried out, no release or degradation of the target compound could be demonstrated during microfiltration (Student's paired test, p-value = 0.38).

[0189] An increase of 10 to 50% in oleacein purity was observed in clarified olive mill wastewater. This is linked in particular to a dry matter retention of between 42 and 65%. The oleacein concentration in clarified olive mill wastewater generally varies from 0.9 to 3.8 g / kg and depends mainly on the type of initial olive mill wastewater.

[0190] At semi-industrial scale, as at pilot scale, olive mill wastewater with a lower total dry matter content continues to be more favorable in terms of flux. Since more heavily loaded olive mill wastewater is likely to have higher initial concentrations of the target compound, a particular embodiment may include a step of blending olive mill wastewater, such as raw olive mill wastewater; for example, a blend of olive mill wastewater from 3-phase (P3) and 2-phase U2 (type P2,5) extraction processes.

[0191] Thus, a mixture of highly loaded olive mill wastewater (type P3 with MS > 10%) with lightly loaded olive mill wastewater (type P2.5 with MS < 6%) advantageously allows standardization of the olive mill wastewater to be filtered in terms of dry matter and oleacein content in order to maximize permeate flux density and oleacein concentration in the permeate. Tangential nanofiltration stage

[0192] For the choice of membrane type, preliminary tests carried out using olive mill wastewater harvested the previous year showed the possibility of developing two types of strategies depending on the type of membrane selected: an enrichment strategy focused on the valorization of the retentate and a purification strategy focused on the valorization of the permeate.

[0193] For this purpose, two flat membranes (SUEZ™) of different selectivities were tested on laboratory scale at filtration pressures ranging from 15 to 30 bar: the DK membrane has an estimated cut-off threshold of 200 Da while it is estimated at 1000 Da for the GE membrane.

[0194] Microfiltered olive mill wastewater is used as the initial juice for nanofiltration, and it is desired to compare olive mill wastewater from Picholine variety olives obtained by 3-phase (P3) and 2-phase U2 (P2,5) extraction. Nanofiltration is carried out at 50 °C.

[0195] Tests without concentration (with a mass reduction factor of 1) were carried out in the laboratory on a pilot with a nominal volume of 3 L and a membrane surface area of ​​0.0125 m2. The objective of these tests is to optimize the operating conditions of each of the two membranes and to determine whether, under these optimal conditions, the performance obtained is compatible with an industrial application. A - Nanofiltration with polyamide-TFC membrane

[0196] The pressure selected following this optimization is 30 bar: this pressure allows flow densities of approximately 50 kg·h⁻²·m² for clarified P3 olive mill wastewater and approximately 75 kg·h⁻²·m² for clarified P2.5 olive mill wastewater. At this working pressure, oleacein is almost entirely retained (98%) in both cases, which is very satisfactory. Indeed, there is no significant difference in retention between clarified olive mill wastewater from P3 and P2.5.

[0197] Total dry matter is also mostly retained (98%) while a small part of the total polyphenols is carried into the permeate (retained at 87%).

[0198] The validation of the process with a DK (Suez™) polyamide-TFC membrane is carried out by confirming that the cumulative flux densities are greater than 10 kg·h⁻², thus compatible with an industrial application. As before, less heavily loaded olive mill wastewater, such as P2.5, is more favorable in terms of flux density. Depending on the type of clarified olive mill wastewater used, a mass reduction factor (MRF) of 2 to 3 can be achieved, which is consistent with typical nanofiltration applications. Table 5 summarizes the characteristics of the filtration products.

[0199] [Tables5] Initial clarified margins, retentate after nanofiltration, concentration factor, FRM ratio, MS (%), PPT (g / kg MF), Oleacein, MS (%), PPT (g / kg MF), Oleacein, MS, PPT, Oleacein, C (g / kg MF), C (g / kg MF), P, MS (g / kg MF), P, PPT (g / 100 gp PT), 2 phases / 2, 5.1 (0.0), 4.8 (0.0), 0.9 (0.0), 12.9 (0.0), 10.6 (0.2), 1.8 (0.0), 13.9 (0.2), 16.9 (0.5), 2.5 (0.0), 1.4 (0.0), 2.1 (0.0), 2.6, 3 phases, 9.0 (0.0), 9.1 (0.1) 1.8 (0.0) 16.3 (0.0) 15.2 (0.4) 2.5 (0.1) 15.2 (0.4) 16.4 (0.8) 1.8 (0.0) 1.7 (0.1) 1.4 (0.1) 1.8

[0200] DM: total dry matter; PPT: total polyphenols; C: concentration; PMS: average purity of oleacein relative to total dry matter; Pppt: average purity of oleacein relative to total polyphenols; MF: fresh matter

[0201] The concentration of oleacein in the final retentate varies from 1.8 to 2.5 g / kg depending on the olive mill wastewater tested with a concentration factor between 1.4 and 2.1. Tangential flow nanofiltration with the DK (polyamide-TFC) membrane can therefore be used to pre-concentrate the extracts.

[0202] Also, under these conditions when microfiltration is combined with nanofiltration, the overall performance of the process is compatible with an industrial application and allows the concentration of the olive mill wastewater by 1.7 to 2.4 times to obtain a mass concentration of approximately 2 g*kg 1 for P2.5 type olive mill wastewater for example.

[0203] B - Nanofiltration with composite polyamide membrane

[0204] The use of a GE membrane (SUEZ™) with a higher cut-off threshold (1000 Da) allows for more pronounced differences between the retentions of the different compounds and thus to move towards purification strategies that allow for a compromise between the lowest possible oleacein retention and the highest possible flux density, dry matter and polyphenol retention.

[0205] With this membrane, the relevant working pressures are between 20 and 25 bar. Under these conditions, the retention of oleacein is generally less than 20% and the retention of dry matter and polyphenols is close to 60% and 50%, which will allow at least partial purification of the target compound.

[0206] At these pressures, flux densities between 25 and 29 kg·h⁻²·m² are achieved for clarified olive mill wastewater from two-phase U2 extraction processes (P2.5) and 21 and 22 kg·h⁻² for clarified olive mill wastewater from a three-phase extraction process (P3). We are therefore in lower ranges than with the DK (polyamide-TFC) membrane, the values ​​remaining compatible with an industrial application.

[0207] The validation of the process with the GE (polyamide composite) membrane was carried out by confirming that the initial flux densities are lower with this type of membrane; however, they decrease less rapidly than with the DK (polyamide-TFC) membrane. Ultimately, the cumulative flux density obtained is 24 kg·h⁻²·m² (therefore greater than 10) and thus remains compatible with industrial application. Under these conditions, it is possible to achieve a FRM close to 3, corresponding to a permeate production yield of 67%.

[0208] Table 6 allows for a comparison of the clarified P2.5 olive mill wastewater before and after nanofiltration: an oleacein purification factor close to 2 is obtained with respect to dry matter and close to 1.5 with respect to purification relative to total polyphenols. In this case, the final permeate has an oleacein purity of approximately 3% relative to dry matter and 27% relative to total polyphenols, which makes the Nanofiltration is a step suitable for the pre-purification and / or purification of the target compound in raw olive mill wastewater:

[0209] [Tableauxô] clarified margins initial permeate after nanofiltration oleacein purification factor FRM MS (%) PPT (g / kg MF) Oleacein MS (%) PPT (g / kg MF) Oleacein MS PPT C (g / kg MF) C (g / kg MF) P MS (g / kg MF) P PPT (g / 100g PPT) 5.2 (0.0) 4.6 (0.0) 0.9 (0.0) 2.7 (0.0) 3.0 (0.0) 0.8 (0.0) 29.5 (2.1) 26.7 (2.1) 1.8 (0.2) 1.4 (0.1) 2.5

[0210] Also, under these conditions when microfiltration is combined with nanofiltration, the overall performance of the process remains compatible with an industrial application and allows to purify about twice the oleacein in olive mill wastewater compared to total polyphenols and a little more than twice compared to total dry matter: for P2.5 type olive mill wastewater for example the final purity of oleacein is 2.7 - 3.5% compared to dry matter and 20 - 27% compared to total polyphenols.

[0211] The use of the GE membrane in a nanofiltration process following microfiltration offers an advantageous pre-purification of raw olive mill wastewater at a lower cost. Furthermore, depending on the technique chosen to finalize the purification of the target compound, the purity of the oleacein relative to the total polyphenols obtained at the end of the process (close to 30% relative to the total polyphenols) is advantageous.

[0212] Tangential flow nanofiltration with DK membrane thus allows a concentration of oleacein (x 3) and a reduction in the quantity / volume of product to be treated ( / 3), reducing the amount of solvent and minimizing the energy expenditure required for any additional treatment steps.

[0213] These data therefore validate the tangential nanofiltration protocol of all the tested olive mill wastewaters, and the industrial application of microfiltration / nanofiltration coupling.

[0214] Example 2: validation of the biological properties of oleacein from olive mill wastewater.

[0215] The work carried out aims to evaluate the anti-aging and antioxidant activity of oleacein produced according to a process of the invention, on the in vivo model of The nematode Caenorhabditis elegans. C. elegans is an excellent model organism for studying aging. Indeed, many major pathways involved in longevity are conserved from the nematode to humans. Among them is the Daf-16 pathway (FOXO in mammals), whose activation is implicated in increased longevity and reduced oxidative stress in C. elegans.

[0216] In humans, FOXO proteins are involved in stem cell maintenance, and their activation also leads to the transcription of genes that protect against oxidative stress. More specifically, FOXO3 is an inhibitor of the NF-κB factor and therefore of inflammation. Exceptional longevity in humans has also been associated with a polymorphism of the FOXO3 gene. Materials and methods C. elegans strains

[0217] All C. elegans strains used were provided by the Caenorhabditis Genetic Center (CGC, University of Minnesota, USA). The daf-16::GFP transgene (strain TJ356) is a C. elegans strain in which the DAF-16 transcription factor is labeled with the fluorescent protein GFP, allowing visualization of its localization within cells (cytoplasmic, intermediate, or nuclear).

[0218] Strain N2 corresponds to the wild-type strain (unmodified). It is a reference strain. Origin of oleacein

[0219] Three types of oleacein are evaluated here: - commercial oil extract (90% - Toronto Research Chemicals) - olive mill wastewater extract obtained according to a process of the invention at 93% - synthetic molecule 99% (University of Tsukuba)

[0220] The effect of the tested preparations on the longevity of C. elegans is compared to that of hydroxytyrosol (> 98% - H4291 Sigma Aldrich). The nematodes are placed directly in contact with oleacein in liquid medium. Results

[0221] Influence of oleacein on the longevity of C. elegans:

[0222] Synthetic oleacein (99% University of Tsukuba) at 5 pg / mL enabled a significant increase in average life expectancy of wild C. elegans (strain N2) of 16% (p-value <0.0001, Kaplan-Meier).

[0223] Oleacein extracted from olive oil (90% Toronto Research Chemicals) at 5 pg / mL resulted in a significant increase in the average life expectancy of wild C. elegans (strain N2) of 22% (p-value <0.0001, Kaplan-Meier).

[0224] Oleacein extracted from Picholine variety olive mill wastewater (93% / Own production) at 5 pg / mL resulted in a significant increase in the average life expectancy of wild C. elegans (strain N2) of 18% (p-value <0.0001, Kaplan-Meier).

[0225] There is no significant difference between these three treatments, which leads to the conclusion that the observed activity of the oil and olive mill wastewater extracts is due to oleacein and that its origin has no impact on activity for similar purities. Oleacein therefore has a positive impact on the longevity of C. elegans; this activity is preserved in the fractions obtained from the processes according to the invention.

[0226] The activity of oleacein was compared to that of hydroxytyrosol (>98% / H4291 Sigma Aldrich), another molecule extracted from olive mill wastewater as a cosmetic ingredient and known for its antioxidant activity.

[0227] Hydroxytyrosol at 5 pg / mL did not increase the average life expectancy of wild C.elegans (strain N2) (p-value = 0.238, Kaplan-Meier).

[0228] Brunetti et al. (2020) previously showed that treatment with hydroxytyrosol at 250 pg / mL allows a significant increase in the average life expectancy of wild C. elegans (strain N2) of 14%.

[0229] These results allow us to conclude that oleacein is more effective (at least 50X more active) than hydroxytyrosol on the longevity of C. elegans.

[0230] Identification of the pathways of action of oleacein on the longevity of C.elegans.

[0231] For these results, oleacein extracted from olive oil (90% / Toronto Research Chemicals) was used. No difference in activity was observed in this experiment compared to oleacein extracted from olive mill wastewater, demonstrating the preservation of oleacein's properties as the main active ingredient after the extraction process from olive mill wastewater. The effect of oleacein at 5 pg / mL on longevity was studied in mutant C. elegans in which the expression of a particular gene was repressed. If the effect observed in the wild type disappears in the mutant, then the repressed gene is involved.

[0232] Oleacein at 5 pg / mL did not increase the average life expectancy of the C. elegans Daf-16 mutant (strain GR1307) (p-value = 0.291, Kaplan-Meier).

[0233] The Daf-16 pathway is therefore involved in the positive effect of oleacein on the longevity of wild C. elegans (strain N2).

[0234] The daf-16::GFP transgene (strain TJ356) is a strain of C. elegans in which the DAF-16 transcription factor is labeled with the fluorescent protein GFP, allowing visualization of its localization within cells (cytoplasmic, intermediate, or nuclear). Activation of the Daf-16 pathway is associated with nuclear translocation of its transcription factor. Pretreatment with oleacein at 5 pg / mL resulted in a significant increase in nuclear translocation. of the transcription factor DAF-16::GFP of 10% (multiplied by 2) compared to the control (p-value < 0.0001, z-test for comparison of proportions).

[0235] Oleacein is therefore an activator of the Daf-16 pathway.

[0236] Activation of the DAF-16 / FOXO transcription factor by oleacein may result from its action on DAF-16 / FOXO partners located upstream in the pathway. The following experiments explore the possibility that activation of the Daf-16 / FOXO pathway by oleacein results from its action on two partners in particular: Daf-2 (IGF-1 in humans) and Sir-2.1 (SIRT1 in humans):

[0237] - Daf-2 / IGF-1 exerts an inhibitory effect on the activity of the transcription factor DAF-16 / FOXO is its primary target. When the activity of the Daf-2 gene is decreased, that of the product of the Daf-16 gene is therefore overactivated.

[0238] - the product of the Sir-2.7 / SIRT1 gene is a modulator of the transcription factor DAF-16 / FOXO is involved in stress resistance. In humans, SIRT1 enhances FOXO's ability to induce cellular resistance to oxidative stress and prevents apoptosis. In the absence of the modulator SIR-2.1 / SIRT1, DAF-16 / FOXO activation by stress induces apoptosis of damaged cells rather than their survival and resistance.

[0239] Oleacein at 5 pg / mL significantly increased the mean lifespan of the C. elegans Daf-2 mutant (strain CB 1370) by 21%, similar to the effect observed in the wild-type strain (p-value < 0.0001, Kaplan-Meier). Therefore, Daf-2 is not involved in the positive effect of oleacein on the longevity of wild-type C. elegans (strain N2). The activation of Daf-16 / FOXO by oleacein is thus independent of Daf-2 (insulin / IGF-independent).

[0240] Oleacein at 5 pg / mL did not increase the mean lifespan of the C. elegans Sir-2.1 mutant (strain VC199) (p-value = 0.454, Kaplan-Meier). Sir-2.1 is therefore involved in the positive effect of oleacein on the longevity of wild-type C. elegans (strain N2). Activation of DAF-16 / FOXO by oleacein is thus linked to the activation of SIR-2.1 / SIRT1.

[0241] Influence of oleacein on thermal and oxidative stress in C.elegans.

[0242] For these results, oleacein extracted from olive oil (90% / Toronto Research Chemicals) was used. No difference in activity was observed with oleacein extracted from olive mill wastewater.

[0243] Heat stress causes an increase in endogenous free radicals in C. elegans, and therefore in situ oxidative stress. A pretreatment with oleacein at 5 pg / mL resulted in a significant increase in the survival rate of wild-type C. elegans (strain N2) of nearly 20% compared to the control after exposure to heat stress of 35°C for 5 hours (p-value < 0.0001, z-test for comparison of proportions). Oleacein therefore protects C. elegans from heat stress and thus from endogenous oxidative stress.

[0244] Adding hydrogen peroxide (H2O2) to the medium causes exogenous oxidative stress in C. elegans. A pretreatment with oleacein at 5 pg / mL resulted in a significant 15% increase in the survival rate of wild-type C. elegans (strain N2) compared to the control after exposure to 0.6 mM H2O2 for 30 minutes (p-value < 0.0001, z-test for comparing proportions). Oleacein therefore protects C. elegans from exogenous oxidative stress.

Claims

Demands

1. A process for treating olive oil pomace, comprising the following steps: a. supplying an olive oil pomace composition, the pomace being characterized by a dry matter content of between 3% and 20% of the total mass of the composition; b. adjusting the pH of the composition to an acidic pH, of between 3.5 and 5.5; c. enzymatic treatment of the adjusted composition resulting in a decrease in the viscosity of the composition by a factor of 2.7 to 2.1 after enzymatic liquefaction; d. treatment of the composition by microfiltration (MF) with a TiO2 ceramic membrane, and collection of the microfiltered fraction; e. treatment of the microfiltered fraction by nanofiltration (NF), and collection of the nanofiltered fraction, characterized in that said process does not comprise a step of separating the solid and liquid phases before the microfiltration step.

2. Process for treating olive oil pomace according to claim 1, the pomace being characterized by a dry mass content of between 4% and 13% of the total mass of the composition.

3. Process for treating olive oil pomace according to any one of claims 1 or 2, the pomace being obtained from a two-phase or three-phase process system.

4. Process for treating olive oil wastewater according to any one of the preceding claims, the wastewater being from the Picholine variety, or from a varietal mixture including the Picholine variety.

5. Process for treating olive oil pomace according to any one of the preceding claims, the enzymatic treatment step being carried out in the presence of one or more pectinolytic enzymes.

6. Process for treating olive oil pomace according to any one of the preceding claims, the microfiltration (MF) step being carried out through a ceramic membrane with an average pore diameter of between 0.1 and 1.4 pm; the microfiltration (MF) step being in particular a tangential microfiltration (MFT) step.

7. A process for extracting oleacein, comprising the implementation of a process for treating olive oil margins according to any one of the preceding claims, and the presence of at least one step of collecting a fraction comprising oleacein; in particular a step of collecting a fraction comprising oleacein from the nanofiltered fraction.

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