FR2981274A1 - PROCESS FOR THE PREPARATION OF COMPLEXES COMPRISING AMYLOSE ASSEMBLED WITH AT LEAST ONE ORGANIC MOLECULE OF INTEREST - Google Patents

Abstract

The present invention relates to a process for the preparation of complexes comprising amylose assembled with at least one organic molecule of interest. This method according to the invention comprises the following steps: (a) a lipophilization step of grafting a fatty aliphatic chain on said organic molecule of interest, for the preparation of a lipophilic derivative of said organic molecule of interest, ( b) a step of bringing into contact between (i) said lipophilic derivative of said organic molecule of interest bearing said grafted fatty aliphatic chain, resulting from said lipophilization step (a), and (ii) said amylose, and (c) a step of forming said amylose / lipophilic derivative complexes of the organic molecule of interest. The present invention also relates to organic chemical compounds in the form of a complex, comprising amylose assembled with at least one organic molecule of interest, in which the said organic molecule of interest is in a lipophilic derivative form, carrying a fatty aliphatic grafted chain.

Description

FIELD OF THE INVENTION The present invention relates to processes for the preparation of complexes in which amylose constitutes the "host molecule", as well as the complexes resulting from these processes.

PRIOR ART Starch is a polysaccharide of plant origin. It is the main source and carbohydrate reserve synthesized by higher plants (cereals, tubers and legumes) during photosynthesis. The starch is native in the form of grains ranging in size from one micron to one hundred microns depending on the botanical origin. Starch consists of two fractions: a very predominant carbohydrate fraction (98 to 99%) and a very minor non-carbohydrate fraction (0.1 to 2%). The carbohydrate fraction of the starch consists mainly of a mixture of two α-D glucopyranosyl polymers, namely amylopectin and amylose, in variable proportions depending on the origin of the starch. Amylose represents about 20 to 30% of the carbohydrate portion of the starches. But this quantity can reach up to 80% for certain varieties of maize (Zea mays), barley (Hordeum vulgare), rice (Oriza sativa) or peas (Pisum sativum). Amylose is a substantially linear molecule composed of 500 to 6000 αD glucopyranosyl units, distributed over 1 to 20 different chains with an average degree of polymerization of 500. The molar mass of amylose is generally between 2.105 and 2.106 g. / mol and depends on the botanical origin but also on the fractionation method used. An amylose population is heterogeneous, with a polydispersity generally between 1.3 and 2.1 but which sometimes reaches higher values (5-10).

Amylose is known for its ability to form stable complexes with other molecules, i.e. a molecular association in which amylose is a "host" molecule (receptor or sequestrant) and the molecule (s) of interest form "guest" molecules (substrate or complexing agent). The links between the "host" molecule and the "guest" molecule are weak interactions (in particular non-covalent bonds). The complexes encountered generally consist of so-called "inclusion" complexes (also called simply "clathrate"). The "guest" molecule is then totally or partially encapsulated in the "host" molecule. More specifically, amylose is likely to complex with hydrophobic molecules (or lipophilic) because of its essentially linear character, and the almost exclusive presence of ct- (1,4) type bonds. This complexation is based on a conformational rearrangement which is induced by the complexing agent and which leads to a new helical conformation in simple helices (6 glucose units per revolution), in which the hydrophilic groups of the chain are turned outwards and the hydrophobic groups inwards. This results in the formation of a hydrophobic helical cavity in the simple amylose helix, which can be occupied by the hydrophobic compounds. This phenomenon of complexation with amylose is used in so-called "molecular encapsulation" techniques.

Molecular encapsulation techniques with amylose enable the protection of a molecule of interest that is normally sensitive to oxidation, hygroscopicity, interaction with other components, light, acidity and alkalinity and / or evaporation. They make it easier to use, mask taste and odor, increase stability and control the release mode (thermal, enzymatic, mechanical).

The formation of complexes with amylose also makes it possible to incorporate and stabilize a hydrophobic molecule in an aqueous medium. This technique can be used for the molecular encapsulation of active ingredients of interest in the pharmaceutical, cosmetic and agri-food fields. A large number of molecules possess this type of complexing property with respect to amylose, in particular the hydrophobic molecules of the fatty acid and fatty alcohols type. But many other molecules of interest are not able to form such complexes with amylose. This is particularly the case for most antioxidants, for example phenolic compounds, especially because of their size, their structure and / or their polarity. Among the natural antioxidants, phenolic compounds, and more particularly phenolic acids, are of growing interest. These molecules, while protecting against oxidation, act against oxidative stress (damage caused in the body by the action of free radicals) and contribute to the prevention of certain diseases such as cardiovascular diseases, cancers, joint problems and neurovegetative diseases. The use of these phenolic compounds in the emulsion-type formulations is delicate (fragile and easily oxidizable molecules) and may be accompanied by a decrease in their effectiveness in protecting the lipids against oxidation, but also contribute to a loss. of their nutritional intake. Indeed, in the aqueous phase are often produced the first free radicals (it is the medium through which diffuses the oxygen coming from the external medium). Moreover, after ingestion, the potential antioxidant power of phenolic compounds vis-à-vis lipids and proteins of the digestive system is likely to be limited or neutralized, by the formation of new interactions with proteins or with the food matrix , at the oral and gastrointestinal level, and especially if the phenolic functions are blocked. In view of the foregoing, it would be particularly advantageous to be able to complex amylose with these molecules of interest, so as to protect them by molecular encapsulation phenomenon. There is therefore a need for novel processes for the preparation of complexes between, on the one hand, amylose and, on the other hand, organic molecules of interest which are not intrinsically suitable for forming complexes with this "host" molecule.

SUMMARY OF THE INVENTION The present invention relates to a process for the preparation of complexes comprising amylose assembled with at least one organic molecule of interest. This method is characterized in that it comprises the following steps: (a) a lipophilization step of grafting a fatty aliphatic chain on the organic molecule of interest, for the preparation of a lipophilic derivative of said organic molecule d interest, (b) a step of bringing into contact between (i) said lipophilic derivative of said organic molecule of interest carrying said grafted fatty aliphatic chain, resulting from said lipophilization step (a), and (ii) said amylose, and (c) a step of forming said amylose / lipophilic derivative complexes of the organic molecule of interest. This strategy consists of considerably reducing the polarity of the molecules of interest by grafting a fatty aliphatic chain. This step makes it possible in particular to give it a complexing action with amylose, advantageously by the imprisonment of the fatty aliphatic chain grafted into the helical cavity of the simple amylose helix thus formed.

In addition, the complexes obtained advantageously do not have any crystalline form (amorphous inclusion complex), which makes it possible in particular to eliminate any precipitation due to the formation of crystallites, which then leads to high instability in the gelled aqueous phase of an emulsion. . The organic molecule of interest is advantageously chosen from polyphenols, more preferably from phenolic acids. In this case, the organic molecule of interest is advantageously chosen from hydroxycinnamic acids or hydroxycinnamic acid esters. The organic molecule of interest in the form of hydroxycinnamic acid is preferably selected from ferulic acids, p-coumaric acids, o-coumaric acids, caffeic acids and sinapic acids.

The organic molecule of interest in the form of hydroxycinnamic acid ester is preferably chosen from among caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and sinapoylquinic acids, or from rosmarinic acids.

In the latter case, the lipophilization step (a) advantageously consists in grafting the fatty aliphatic chain onto the quinic group of the organic molecule of interest, more preferably on one of the hydroxyl functional groups of the quinic group of the organic molecule. interest.

In particular, the organic molecule of interest advantageously consists of 5-O-coffeeoylquinique; the lipophilization step (a) consists in grafting the fatty aliphatic chain on the hydroxyl group in position 3 or in position 4 of the quinic group of said organic molecule of interest.

Other advantageous characteristics, which can be taken independently or in combination, are developed below: the lipophilic derivative of the organic molecule of interest, resulting from the lipophilization step (a), comprises a fatty aliphatic grafted chain comprising (see consisting) a linear (unbranched) alkyl group, having a chain length of preferably between 12 and 16 carbon atoms, inclusive; the step of forming the complexes (c) is carried out so as to obtain inclusion complexes (advantageously amorphous), in which the fatty aliphatic chain of lipophilic derivatives of organic compounds of interest is included in one of helices of the associated amyloidosis; during the contacting step (b), the amylose molecule consists of amylose alone or of amylose in the form of starch. The present invention also relates to an organic chemical compound in the form of a complex, comprising amylose assembled with at least one organic molecule of interest; this organic molecule of interest is in a lipophilic derivative form, carrying a fatty aliphatic grafted chain. This organic chemical compound is advantageously obtained by carrying out the process developed above. The organic molecule of interest bearing the grafted fatty aliphatic chain is as defined above. In particular, this organic molecule of interest is advantageously chosen from among caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and sinapoylquinic acids, or from rosmarinic acids.

In this case, the lipophilic derivative of the organic molecule of interest advantageously carries the fatty aliphatic chain grafted on its quinic group, preferably on one of the oxygens covalently bonded to the cyclohexyl group of the quinic group. Preferably, the lipophilic derivative of the organic molecule of interest consists of a 5-O-caféoylquinique, whose grafted fatty aliphatic chain is carried by the oxygen atom in the 3-position or in the 4-position of the quinic group. The grafted fatty aliphatic chain advantageously comprises (see consists of) a linear (unbranched) alkyl group, the chain length of which is advantageously between 12 and 16 carbon atoms, inclusive. The organic chemical compound is advantageously in the form of an inclusion complex (advantageously amorphous), in which the fatty aliphatic chain grafted lipophilic derivatives of organic compounds of interest is included in one of the helices of the associated amylose. DESCRIPTION OF THE FIGURES Figure 1: differential scanning calorimetry ("AED") thermogram of amylose alone; Figure 2: AED thermogram of the amylose / chlorogenic acid assembly: (A) mounted 1, (B) descent, (C) mounted 2; Figure 3: AED thermogram of the amylose / hexadecyl chlorogenate assembly: (A) mounted 1, (B) descent, (C) mounted 2; 4: AED thermogram of the amylose / 3-0-chlorogenic palmitoyl acid assembly: (A) mounted 1, (B) down, (C) mounted 2; FIG. 5: AED thermogram of the amylose / 4-0-palmitoyl chlorogenic acid assembly: (A) mounted 1, (B) descent, (C) mounted 2; Figure 6: X-ray diffraction pattern for (A) amylose alone, (B) amylose / chlorogenic acid assembly, (C) amylose / 4-chloro-palmitoyl acid chlorogenic assembly, (D) amylose / chlorogenic 3-0-palmitoyl acid assembly and (E) the amylose / hexadecyl chlorogenate assembly; Figure 7: Solid state NMR 130 spectrum (CP-MAS) for (A) amylose alone, (B) amylose / chlorogenic acid assembly, (C) amylose / 4-0-palmitoyl acid assembly chlorogenic, (D) the chlorogenic amylose / 3-0-palmitoyl acid assembly and (E) the amylose / hexadecyl chlorogenate assembly; Figure 8: Schematic model of the complex amylose / acid 4-0chlorogenic acid.

DETAILED DESCRIPTION OF THE INVENTION The present invention thus provides a new process for the preparation of complexes comprising amylose encapsulating at least one organic molecule of interest, the latter not being initially capable of forming such complexes with amyloidosis. In particular, this invention relates to a process for preparing complexes in which the following steps are carried out: (a) a lipophilization step of grafting a fatty aliphatic chain onto said organic molecule of interest, for the preparation of a derivative lipophilic of said organic molecule of interest, (b) a step of bringing into contact between (i) said lipophilic derivative of said organic molecule of interest bearing said grafted fatty aliphatic chain, resulting from said lipophilization step (a), and (ii) said amylose, and (c) a step of forming said amylose / lipophilic derivative complexes of the organic molecule of interest. Organic Molecule of Interest The organic molecule of interest advantageously consists of a bioactive molecule. This organic molecule of interest, in the native state (before the lipophilization step), is not capable of forming a complex with amylose (molecule in the native state non-complexable with amylose).

In particular, this organic molecule of interest advantageously does not include, intrinsically, a group or radical (preferably a side chain, advantageously a linear alkyl side group) capable of lodging in a simple helix of amylose. The organic molecule of interest is advantageously chosen from among molecules having an antioxidant activity, more preferably from polyphenols. By "antioxidant activity" is advantageously meant a molecule which decreases, or prevents, the oxidation of other chemical substances. A molecule capable of neutralizing or reducing the damage caused by free radicals in the body is still advantageously meant. By "polyphenols", or "phenolic compounds" is meant in particular organic molecules having one or more aromatic rings (or benzene rings), bearing one or more hydroxyl functions.

All plants contain such phenolic compounds but, as is the case for most natural substances described as secondary metabolites, their qualitative and quantitative distribution is unequal according to species, organs, tissues and physiological stages. Phenolic compounds are known for various actions: anticarcinogen for some of them, and antioxidant to fight against cellular aging. The phenolic compounds can be grouped into classes that are differentiated first by the complexity of the basic skeleton (ranging from a simple C6 to extremely complex highly polymerized forms), then by the degree of modification of this skeleton (degree of oxidation, etc.), finally by the possible links of these basic molecules with other molecules (carbohydrates, lipids, proteins, etc.). Thus, in the case of such phenolic compounds, the organic molecule of interest is advantageously chosen from phenolic acids.

A phenolic acid (or "phenol acid") is an organic compound having at least one carboxylic function and one phenolic hydroxyl. By "phenolic acid" is meant in particular: (i) compounds derived from benzoic acid, namely hydroxybenzoic acids, and (ii) compounds derived from cinnamic acid, namely hydroxycinnamic acids or esters of hydroxycinnamic acids. The hydroxybenzoic acids have the following formula (I): ## STR2 # R2 = OH. Hydroxybenzoic acids are derived from benzoic acid and have a basic formula of the C6-C1 type, of which the most abundant in the plant kingdom are the gallic, protocatechic, vanillic and syringic acids. The hydroxycinnamic acids for their part have the following formula (II): ## STR1 ## in which Ade.terulic Adde p-coconutic Adde.o-coumahque Ac caligale Acid shapciiie Rf = 0H 3: = = H, R2 = 0H R4 = 0H I = R2 = OH R3 = R4 = h1 = R3 = OCH: 3 'R-7 = 0H,: R4 = H These hydroxycinnamic acids represent a very important class whose basic structure (C6-C3) derives from that of cinnamic acid. In this subdivision, caffeic, ferulic, p-coumaric and sinapic acids are found in particular. The esters of hydroxycinnamic acids (or hydroxycinnamates) generally comprise a glucose group, quinic acid or tartaric acid. The family of hydroxycinnamates includes in particular the esters of caffeic, ferulic and p-coumaric acids with quinic acid, but also rosmarinic acid, which is a particular ester of caffeic acid and 3- (3, 4) -dihydroxyphényl-lactic acid.

The organic molecule of interest advantageously consists of "chlorogenic acid" as such, that is to say the ester of caffeic acid and (L) -quinic acid 3 (referred to as trans -5-O-caffeoyl-Dquinate, or caffeylquinic acid, or 5-ACQ (or "5-CQA" in English) or 5O-caffeylquinique).

The formula (III) of chlorogenic acid is as follows: ## STR1 ## By "chlorogenic acid" is also meant the family of acids whose chemical structure is similar to the abovementioned chlorogenic acid, and which groups the compounds formed by one or more hydroxycinnamic acid conjugated with quinic acid, The organic molecule of interest thus encompasses the compounds formed of quinic acid conjugated hydroxycinnamic acids, forming the "class (or family chlorogenic acids whose formula (IV) is the following: ## STR2 ## in which: R 1 = R 2 = OH, R = R 4 R 1 = = H, R 1 = OCH: 7-1, R 2 = OH, R 3 = HP, I = R2: = OCH3, R4 = H .A of cataeaylquinkiue .Addep-coumaroylqunicique Adde'fndoylquinkiue.Adde sinapayique: irdque Thus, by "family of chlorogenic acids", we mean the mono-esters, in particular the caffeylquinic acid (ACQ), ferulylquinic acid (FFA), p-coumarylquinic acid (QPA), sinapylquinic acid, dimethic acid oxycinnamylquinique.

The term "chlorogenic acid family" also refers to di-esters, namely in particular dicaféylquinique acid, caféylférulylquinique acid, diférulylquinique acid, di-p-coumarylquinic acid and dimethoxycinnamylquinique acid. By "family of chlorogenic acids" is meant finally the isomeric forms. For example, depending on whether the bond is at the 3, 4 or 5 position on the hydroxyls of quinic acid, the following are included: 3-O-Caffeylquinic, 4-O-Caffeylquinic and 5-O-Caffeylquinic, for cafylquinic acids (ACQ); 3-O-ferulylquinic, 4-O-ferulylquinic and 5-O-ferulylquinic for ferulylquinic acids; and 3-O-p-coumarylquinic, 4-O-p-coumarylquinic and 5-O-pcoumarylquinic, for p-coumarylquinic acids. Compounds consisting of several hydroxycinnamic acids conjugated with a quinic acid, such as 1,3-dicaffeylquinic acid, 3,4-o-dicafeylquinic acid, 3,5-o-dicafeylquinic acid, etc., are also encountered. coffeeylferulylquinic acid. The rosmarinic acid has the following formula (V): (V) On the lipophilization step The lipophilization step (a) has the objective of functionalizing the organic molecule of interest so that to give it properties of a complexing agent with amylose. By "lipophilization" is meant the chemical reaction of yielding, by a covalent bond, a fatty aliphatic chain on a receptor moiety of the organic molecule of interest. The grafted fatty aliphatic chain (or fatty aliphatic side chain), referred to the organic molecule of interest, comprises (advantageously consists of) advantageously an acyclic, linear, non-branched, saturated organic (advantageously hydrocarbon) organic group (or radical). More preferably, this grafted fatty aliphatic chain comprises, or even consists of, a linear non-branched saturated fatty chain, advantageously also an unbranched linear alkyl group (formula CnH2n + 1). This grafted fatty aliphatic chain advantageously comprises a carbon chain length chosen from 12, 14, 16, 18, 20 or 22 carbon atoms, and more preferably chosen from 12 (lauryl or dodecyl group), 14 (myristyl or tetradecyl group). ) or 16 (palmityl or hexadecyl group) carbon atoms. This lipophilization step (a) is chosen from the protocols making it possible to obtain an organic molecule of interest to which a single fatty aliphatic chain is reported by a covalent bond. In the present description, the product resulting from this lipophilization operation is referred to as the "lipophilic derivative" (or "lipophilic derivative" or "lipophilized derivative") of the organic molecule of interest.

For organic molecules of interest belonging to polyphenols, we will speak of "lipophilic derivatives of polyphenols". The processes for lipophilizing polyphenols, by grafting fatty aliphatic chains, are known from the state of the art. Reference can be made in particular to the following documents: Figueroa-Espinoza and Villeneuve, 2005 (J. Agric., Food Chem., 53: 2779-2787); Villeneuve et al. 2007 (Biotechn Adv 25: 515-536), Lopez et al., 2007 (Enz Microb., Tech., 41: 721-726; 01 Fatty Fat Body, 14: 51-59). These synthetic reactions of such lipophilic derivatives of polyphenols are advantageously acylation or alkylation reactions.

The "acylation" reaction may be defined as the establishment of a covalent bond between an acyl group and a nucleophilic function such as a hydroxyl function. The "alkylation" reaction can be defined as a chemical reaction consisting of the transfer of an alkyl group from one organic molecule to another. The synthesis reaction, acylation or alkylation, is chosen according to the nature of the polyphenol, in particular whether or not it has a hydroxyl or carboxyl function (group of phenolic acids or flavonoids and lignans). Thus, in practice: if the acyl donors are fatty alcohols or fatty alcohol esters and the lipophilization takes place on phenolic acids, the reaction will be called the "alkylation" reaction; If the acyl donors are fatty acids or esters of fatty acids and the lipophilization takes place on other polyphenols and in particular on the glycosylated portions of the flavonoids, the reaction will be called an "acylation" reaction . The presence of a quinic portion bearing non-phenolic hydroxyls and a carboxyl group (as well as a caffeic part having phenolic hydroxyls) makes chlorogenic acid one of the rare polyphenols which makes it possible to envisage the synthesis of derivatives by alkylation and also by acylation. The alkylation reaction is described, for example, in Lopez-Giraldo et al., 2007 (OCL 2007, 14: 51-9, and Enz Micro Tech 41: 721-6). The enzymatic synthesis reaction by acylation is, for example, described in the document Lorentz et al., 2010 (Biotechnol Lett 32: 1955-1960). These lipophilization reactions of polyphenols are known to allow the synthesis of molecules having not only antioxidant activities, but also pharmacological properties, surfactant activities, or antimicrobial activities, corresponding to the concept of multifunctional compounds. This lipophilization can be carried out by a chemical synthesis route or by an enzymatic synthesis route, which are conventional in themselves. The protocol is chosen according to the organic molecule of interest to be functionalized, taking into account in particular the desired receptor group as developed after. Chemical Synthesis Several methods have been described for the chemical synthesis of lipophilic derivatives of organic compounds, especially lipophilic derivatives of polyphenols. This chemical synthesis may, for example, be selected from: (i) acylation of the organic molecule of interest by fatty acid chlorides in anhydrous solvents (Zhong and Shahidi, 2011, J. Agric. Food, Chem 59 (12) pp. 6526-6533, Fukami et al., 2008, US-20080058409, Xiang and Ning, 2008, Swiss Food, Inc., Sci., Tech., 41: 1189-1203); (ii) the acylation of the organic molecule of interest by fatty acids and fatty alcohols (saturated, monounsaturated, branched or unbranched) on the hydroxyl and carboxyl functions with or without a catalyst (for example N, N ' dicyclohexylcarbodiimide (DCC) and / or thionyl chloride (50C12)). On the other hand, regioselective coupling reactions with ketones have also been described (Poaty et al., 2009, European Food Research and Technology 230: 111-117). In general, the main drawbacks of the chemical methods are the production of by-products, the use of toxic solvents or catalysts and the production of mixtures of esters or isomers bearing a variable number of solvents. carbon chains that must be separated. In addition, the grafting sometimes involves hydroxyls responsible for the antioxidant activity (use of fatty acid chloride). The methods of protecting and deprotecting hydroxyl groups are also widely used to allow the choice of the reaction site and the degree of esterification but these are often complex and time-consuming steps. Enzymes, on the other hand, are a valuable synthesis tool for mild reaction conditions and regioselectivity. In addition, the increasing demand for products of therapeutic, agri-food or cosmetic interest, in parallel with the requirements for environmentally friendly synthesis processes, reinforces the interest of the use of biocatalysts. In fact, enzymes can replace chemical catalysts and meet the requirements of sustainable industrial development. They can increase production efficiency and reduce energy consumption for those operating at room temperature. Many industrial processes operate at high temperature or high pressure or under highly acidic or basic conditions. At the industrial level, the advantages of using enzymes, despite their sometimes high cost, are simplified purification steps, as well as mild experimental conditions avoiding the degradation of substrates and allowing the reuse of the biocatalyst (Villeneuve, 2007). Biocatalysis makes it possible to envisage the selective production of complex and original structures. For the lipophilization of organic molecules of interest belonging to polyphenols, various enzymes can be used, such as lipases, transferases, isomerases, esterases and proteases. However, lipases are the most frequently used for this type of biotransformation (Figueroa-Espinoza and Villeneuve 2005, Chebil et al., 2006, J. Agric. Food Chem 55: 9496-9502). By "lipases" is meant the enzymes triacylglycerol hydrolases, class EC 3.1.1.3 (according to the classification INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY), belonging to the family 25 of carboxylic ester hydrolases. The lipase advantageously comes from a microorganism (bacterium or fungus), such as Aspergillus piger, Candida rugosa, Candida lipolytica, Rhizopus oryzae, Penicillium camembertii, Penicillium roquefortii and Mucor javanicus. This enzyme can also come from plants or animals. The lipophilization step (a) of the process according to the invention may in particular be carried out with one of the following enzymes.

Novozym 435 is an industrial preparation produced by Novozymes (Dk) and marketed by Sigma, resulting from the immobilization of the B isoform of Candida antarctica lipase on a macroporous acrylic resin. This preparation is in the form of white microbeads with a diameter of between 0.3 and 0.9 mm. It is provided with an activity of 7000 U / g (1 unit is defined as the amount of enzyme which allows the conversion of a pmol of lauric acid into propyl laurate per minute under standard and equimolar conditions). Its water content is 1 to 2% (w / w) and its enzymatic protein content is between 1 and 10%.

Lipozyme RM is a preparation produced and marketed by Novozymes (Dk) resulting from the immobilization of Mucor miehei lipase on a macroporous anion exchange resin. This preparation is in the form of brown microbeads with a diameter of between 0.2 and 0.6 mm. It is provided with a minimum activity of 42 U / g (1 unit corresponds to the amount of enzyme which allows the release of one pmole of oleic acid per minute at pH 8.0 at 40 ° C during hydrolysis triolein). The lipase of Pseudomonas cepacia immobilized on diatomaceous earth is a preparation produced by Amano Enzymes (UK) and marketed by Sigma. This preparation is in the form of beige microbeads and is provided with a minimum activity of 500 U / g (1 unit corresponds to the amount of enzyme which allows the release of one pmole of oleic acid per minute at pH 8.0 at 40 ° C during hydrolysis of triolein). Lipozyme TL is a preparation produced and marketed by Novozymes (Dk) resulting from the immobilization of Thermomyces lanuginosa lipase on silica beads. This preparation is in the form of white microbeads. In general, for a phenolic acid ester, the lipophilization step (a) of the "alkylation" type is advantageously carried out by direct esterification from fatty alcohol in the presence of Novozym 435 (Candida antarctica lipase acrylic resin - SIGMA), with or without a solvent.

The ability of Novozym 435 to catalyze the alkylation of chlorogenic acid with saturated and unsaturated fatty alcohols of varying chain lengths (C8-C18) was investigated. Indeed, chlorogenic acid esters have been synthesized by direct esterification (Guyot et al., 2000, Eur J Sci Technol 102: 93-96) and transesterification (Lopez-Giraldo et al. The bioconversion and the reaction time are improved by means of a chemo-enzymatic strategy which consists of a methylation of the chlorogenic acid followed by a transesterification of methyl chlorogenate in a molten medium (Lopez-Giraldo et al. al., 2007). In the context of such an alkylation reaction, the alcohol donor advantageously consists of a saturated, linear (unbranched) fatty alcohol, the carbon chain length of which is preferably chosen from C12, C14 or C16.

By "acylation reaction" is meant in particular (i) a transacylation reaction or (ii) a direct acylation reaction. For a "transacylation" reaction, the acyl donor is advantageously chosen from fatty acid esters, preferably either methyl or ethyl esters, or vinyl esters.

More preferably, these fatty acid esters comprise a carboxyl group consisting of a linear saturated fatty chain whose length is preferably selected from C12, C14 or C16. These fatty acid esters are thus advantageously chosen from ethyl laurate (C12), ethyl palmitate (C16) and ethyl stearate (C18). For a "direct acylation" reaction, the acyl donor is advantageously chosen from linear saturated fatty acids, and in particular lauric acid (C12), myristic acid (C14) and palmitic acid (C16). . Such biosynthetic reactions are further detailed hereinafter in the Example section, for obtaining three lipophilic derivatives of chlorogenic acid, namely hexadecyl chlorogenate (obtained by alkylation), and 4-0- chlorogenic palmitoyl and chlorogenic 3-O-palmitoyl (obtained by acylation).

On the lipophilic derivative of the organic molecule of interest The lipophilic derivative of the organic molecule of interest, obtained at the end of the lipophilization step (a) described above, comprises a core constituted by the organic molecule of interest described above, carrying a fatty aliphatic side chain grafted.

The lipophilic derivative of the organic molecule of interest thus advantageously carries a single fatty aliphatic side chain. By "side chain" is meant a part of molecule attached to the core or the main chain of the structure, that is to say to the organic molecule of interest.

The grafted fatty aliphatic chain (or fatty aliphatic side chain), referred to the organic molecule of interest, advantageously comprises (optionally consists of) an acyclic organic group (or radical), linear (not connected), saturated. More preferably, this fatty aliphatic grafted chain comprises, or even consists of, a linear non-branched saturated fatty chain, advantageously a linear alkyl group. This grafted fatty aliphatic chain preferably comprises a carbon chain length chosen from 12, 14, 16, 18, 20 or 22 carbon atoms, and more preferably chosen from 12 (lauryl or dodecyl group), 14 (myristyl or tetradecyl group). ) or 16 (palmityl or hexadecyl group) carbon atoms. According to a particular embodiment, the organic molecule of interest in the form of hydroxycinnamic acid ester is chosen from among caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and sinapoylquinic acids, or from rosmarinic acids.

For organic molecules comprising a quinic group (advantageously caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and sinapoylquinic acids), the lipophilic derivative of such an organic molecule of interest advantageously carries the fatty aliphatic chain grafted on its quinic group. In this case, the grafted fatty aliphatic chain is preferably carried by one of the oxygens covalently bonded to the cyclohexyl group of the quinic group. More preferably, the lipophilic derivative of the organic molecule of interest consists of a 5-O-caféoylquinic core (corresponding to the chlorogenic acid molecule), whose grafted fatty aliphatic chain is carried by the oxygen atom in the 3-position. or in position 4 of the quinic group. The lipophilic derivative of such an organic molecule of interest is thus chosen from chlorogenic 3-O-lauroyl acid, chlorogenic 3-O-myristoyl acid, chlorogenic 3-O-palmitoyl acid and 4-chlorogenic acid. Chlorogenic lauroyl, chlorogenic 4-O-myristoyl acid or chlorogenic palmitoyl 4-O-acid. For example, for a C16 fatty aliphatic side chain, the lipophilic derivative of the organic molecule of interest is advantageously a chlorogenic 3-O-palmitoyl acid or a chlorogenic 4-palmitoyl acid. The chemical formula (VI) of chlorogenic 4-O-palmitoyl acid is given below: ## STR2 ## The chemical formula (VII) of 3-O-palmitoyl acid As for the chlorogenic acid, it is recalled below: Among the rosmarinic acids, the lipophilic derivatives of such organic molecules of interest advantageously have the following formula (VIII): HO OH (VIII) in which R, corresponding to the grafted fatty aliphatic chain, comprises (or consists of) an alkyl group (formula CnH2n + 1) whose length of the carbon chain is advantageously chosen from C12 (dodecyl rosmarinate), C14 (tetradecyl rosmarinate) or C16 (hexadecyl rosmarinate) The lipophilic derivatives of rosmarinic acid are for example obtained by catalysis using a lipase Candida antarctica B (i) direct esterification or (ii) transesterification by chemo-enzymatic route. another mode of reality In particular, the organic molecule of interest is chosen from hydroxycinnamic acids, and its lipophilic derivative advantageously has the formula (IX) below: R 5 (IX) in which the organic molecule of interest consists of: R2-OH R4- R3-R4-11 R3-R7'-OCH3., R2-0H, R4-H Folic acid: ligue p-coumadque acid O-cournaric acid Aude cafélque Sinapic acid and in which R5, corresponding to the fatty aliphatic chain grafted, comprises (or consists of) an alkyl group (formula CnH2n + 1) whose length of the carbon chain is advantageously chosen from C12, O14 or O16. According to yet another particular embodiment, the organic molecule of interest is chosen from the hydroxybenzoic acids, and its lipophilic derivative advantageously has the formula (X) below: COOR4 (X) in which the organic molecule of interest consists of: = OH .Adde: gathgue Ri-H, R = OCH. Protocatechic acid RI-Ra-OCH3 Reactive acid Ri = -R3-.H, R2 = 01-1. Acid sWineue p-Hydronbenzffigue acid and wherein R4, corresponding to the grafted fatty aliphatic chain, comprises (or consists of) a alkyl group (formula CnH2n + 1) in which the length of the carbon chain is advantageously chosen from C12, O14 or O16.

On Amylose The complexes according to the invention can be directly prepared from starch. However, the complexes according to the invention are advantageously prepared from amylose alone, especially if it is desired to overcome the presence of amylopectin. The amylose can be extracted from starch grains dispersed in water by selective reprecipitation in the presence of alcohol (butanol for example).

Amylose can also be synthesized in vitro enzymatically either from ADP-glucose with 1,4-α-D-glucan 4-a-Dglucosyltransferase (Granule Bound Starch Synthase, GBSS), or from sucrose with a recombinant amylosaccharase which makes it possible to obtain highly crystalline 35-58 DP amylose.

1,4-α-D-glucan: ortophosphate, α-glucosyl transferase (starch phosphorylase) is also used to produce amylose from glucose-1-phosphate. Amylose can still be obtained from starch with a debranching enzyme: isoamylase or pullulanase.

The characteristics of amylose for the formation of such complexes are further specified in Godet et al., 1995 (Carbohydrate Polymers 27: 47-52).

Contacting step and step of forming the complexes The step of contacting and forming the complexes is advantageously carried out according to a conventional protocol per se. For example, the contacting and forming steps consist of a hydro-thermal stirring protocol as described for example in the Example section below. More specifically, amylose complexes can be prepared from starch. During the cooling following the gelatinization, the addition of the lipophilic derivative of the organic molecule of interest results in the formation of the complexes from the dispersed amylose. The amylose complexes are still advantageously prepared from amylose alone. In this case, the formation of a large number of amylose complexes requires a prior dispersion of the amylose in an aqueous phase (advantageously to form a hydrocolloid) followed by the addition of the lipophilic derivative of the organic molecule. interest, then a precipitation. The dispersion of the amylose can be carried out in a cold solution of sodium hydroxide followed by dialysis against water or neutralization (Buléon et al., 1990). It can also be carried out under hot conditions (higher than 160 ° C.) (Biais et al., 2006) or in the presence of DMSO (Biliaderis et al., 1985, Karkalas et al., 1995, Godet et al. 1993,1995). The addition of the lipophilic derivative of the organic molecule of interest in the gelatinized starch or in the amylose dispersion is generally carried out at a temperature of between 90 ° C. and 100 ° C. Amylose / lipophilic derivative complex of the organic molecule of interest The organic chemical compound in the form of a complex, obtained at the end of the preparation process developed above, comprises amylose assembled with the lipophilic derivative of the molecule. organic compound of interest or with the lipophilic derivative of at least two organic molecules of different interest. This organic chemical compound thus consists of a complex in which amylose constitutes a "host molecule" (receptor or sequestering agent), and the associated lipophilic derivatives correspond to "guest molecules" (substrate or complexing agent). Amylose thus ensures the molecular encapsulation of the lipophilic derivative of the organic molecule of interest. The organic chemical compound obtained advantageously consists of an inclusion complex (also called simply "clathrate"), in which the fatty aliphatic chain grafted lipophilic derivatives of the organic molecule of interest is included in one of the propellers of the amyloidosis associated. Preferably, this inclusion complex is amorphous, or predominantly amorphous. The absence, or near-absence, of crystalline form makes it possible in particular to eliminate the risk of possible precipitation due to the formation of crystallites, which then leads to high instability in the gelled aqueous phase of an emulsion. The characterization of the amylose / lipophilic derivative assemblies of the organic molecule of interest is advantageously carried out by three different techniques, namely differential scanning calorimetry (DSC), X-ray diffraction (XRD) and solid NMR. The formation of an inclusion complex is advantageously demonstrated, on the one hand, by the presence of the fusion endotherm in differential enthalpy analysis and by the reversibility of the formation during cooling, and on the other hand on the other hand, by the presence of an intense singlet at 102.6 ppm in solid NMR. In practice, the Example below demonstrates that the lipophilic derivatives of chlorogenic acid are capable of forming such complexes (advantageously inclusion complexes) with amylose. These results are entirely transposable to any other organic molecule of interest as defined above. For each of these organic molecules of interest, it is sufficient to determine the optimal receptor group for the attachment of the fatty aliphatic chain, and possibly the length of the latter, which allows the complexation of this lipophilized derivative with amylose. The organic chemical compound in the form of a complex can be isolated in a solid form, for example a powder (if precipitation) or a film (for a gel) (in particular depending on the temperature used to solubilize the amylose, and formation of amorphous or crystalline complexes). These solid forms are for example described in Doublier et al., 1989 (Carbohydrate Research 193: 215-226). Composition incorporating the organic chemical compound The chemical compound in the form of a complex according to the invention is advantageously intended to be incorporated in a composition, for example a food composition, a pharmaceutical composition or a cosmetic composition. The chemical compound in the form of a complex may be (i) prepared beforehand and then introduced into the composition or (ii) prepared directly within this composition. This composition consists, for example, of an emulsion (the chemical compound in the form of a complex is then advantageously found in the aqueous phase), an aqueous solution or a hydrogel. This composition may also consist of a solid medium, for example a food product. For example, the composition according to the invention, containing the organic compound in complex form, can be introduced into a food or can consist of a food. The invention is particularly applicable to compositions containing amylose, for example pasta or bakery products. The organic compound in complex form can be incorporated at a suitable time during the preparation of the composition. Alternatively, the organic compound can be formed directly within and during the method of preparing the food composition.

The chemical compound according to the invention can also be used in a pharmaceutical application or a cosmetic application, alone or in a composition containing in particular any other desired active substance and / or appropriate excipent.

Interest of the formation of complexes with amylose The formation of complexes with amylose makes it possible to ensure the molecular encapsulation of the organic molecules of interest associated.

Such a molecular encapsulation makes it possible in particular: to protect the organic molecules of interest against oxidation, to envisage a release of the organic molecules of interest, while preserving the fatty aliphatic chain within the amylose helix by enzymatic digestion, in particular under the action of pancreatic amylases or intestinal lipases, or by acid attack (during digestion or predigestion), - to mask the taste of the organic molecules of interest, - to guarantee the stability of the organic molecules of interest, or - to increase the bioavailability of these organic molecules of interest. In particular, oxidation is probably one of the major parameters at the origin of the deterioration of food products. The resulting oxidative degradations affect the nutritional and sensory qualities of the products and may have repercussions on the health of the consumer. The promotion of antioxidants of plant origin for food is a major challenge for research and industry. The molecular encapsulation of chlorogenic acid, see other phenolic compounds, by amylose has the particularity of ensuring on the one hand, the protection of the ligand against oxidation and, on the other hand, to consider a targeted release of the organic molecule of interest by enzymatic digestion (under the action of pancreatic amylases or intestinal lipases) or under the action of an acid attack.

EXAMPLE Materials and Methods Biological and Chemical Materials The lipase used is Lipozym RM from M. miehei immobilized on an ion exchange resin and purchased from NOVOZYMES (Dk). All solvents and reagents used were obtained from commercial sources and were either HPLC grade or analytical grade. Vinyl palmitate and silica gel plates were purchased from Sigma-Aldrich, France. Chlorogenic acid was purchased from Acros, France, with a purity of 99%. The amyloidosis of potato (type III), essentially free of amylopectin, was obtained from Sigma (France) and used as received. Enzymatic synthesis of acylated lipophilic derivatives of chlorogenic acid Two lipophilic derivatives of chlorogenic acid, namely chlorogenic 3-0-palmitoyl acid and chlorogenic 4-O-palmitoyl acid, were synthesized enzymatically according to a procedure. similar process to that described in Lorentz et al., 2010, supra, with some modifications.

For this, chlorogenic acid, vinyl palmitate and Lipozym RM were dried for at least two days under P2O5, before use.

The activity of the water (aw) of the reaction medium was determined using an AqualabLite® device (Decagon Devices Inc, USA). The initial water activity of all reaction media was below 0.2.

Acylation reactions were performed in 50 ml Eppendorf tubes in the dark. The reaction mixture has a molar ratio (vinyl palmitate / chlorogenic acid) of 40 in 10 mL of 2-methyl-2-butanol (2M2B). The initial concentration of chlorogenic acid was 28 mM.

The vinyl palmitate and the chlorogenic acid were first solubilized for 12 h in 1 mL of 2M2B and stirred at 1000 rpm at 60 ° C in an Eppendorf Thermomixer device (Roucaire, Courtabeuf, France). The reaction was initiated by addition of 40 g / L of enzyme and 200 g / L of molecular sieves (3A), previously dried overnight at 200 ° C. 2M2B was also previously dried on molecular sieves (3A). Samples were removed at different times to monitor the reaction by thin layer chromatography ("TLC" or "TLC" in English) and high performance liquid chromatography ("HPLC"), and purified as described in Lorentz et al. . (2010).

At the end of the synthesis, the immobilized enzyme is separated from the synthesis medium by filtration through a glass filter (1.6 μm). The purification of the products is carried out by chromatography, either on a preparative thin layer (TLC) or on a column or on silica gel or Sephadex. For example, the reaction media, previously filtered in order to remove the enzyme, are concentrated by evaporation under reduced pressure and then deposited on a 20 x 20 cm preparative silica plate. The plate is developed using an eluent and then revealed with UV or iodine vapors in order to locate the migration zone of the synthesized compound. The silica located in this zone is then detached from the support, and the compound is then desorbed in 5 ml of a methanol / chloroform mixture (50/50 v / v) with stirring at 250 rpm for 10 min, this twice. The mixture is then filtered and then evaporated to dryness.

Enzymatic Synthesis of the Alkyl Lipophilic Derivative of Chlorogenic Acid The enzymatic synthesis of hexadecyl chlorogenate was carried out according to the conditions developed by Lopez-Giraldo et al. (2007). In the absence of light, 1.5 g of chlorogenic acid and 260 g of hexadecanol are introduced into a flask for 2 h in a water bath at 55 ° C. and with stirring at 250 rpm in order to allow solubilization. chlorogenic acid in hexadecanol. The reaction is started by the addition of Novozym 435, previously equilibrated for 15 days with a salt solution saturated with NaOH at a theoretical value of 0.05. The amount of catalyst used is 5% (w / w) (calculated on a weight basis taking into account all the substrates involved). In order to direct the reactions towards the synthesis, the water produced by the reaction is adsorbed on a 4 A molecular sieve (30 mg / ml of reaction medium). The purification of the hexadecyl chlorogenate was carried out by column chromatography on silica gel. The method is that described by Lopez-Giraldo et al. (2007). A first liquid-liquid extraction separation step is carried out using 500 ml of a hexane / acetonitrile mixture (1/3 v / v). The hexane phase, which contains the majority of the fatty alcohol, is removed, while the acetonitrile phase, mainly containing the ester, is washed a further 3 times with 100 ml of hexane. After these washes, the acetonitrile phase is evaporated at 40 ° C under reduced pressure using a rotary evaporator. The dry residue is dissolved in 60 ml of a toluene / ethyl acetate mixture (70/30, v / v) and deposited on a glass column (40 × 2.5 cm) of silica (previously dried at 100 ° C. C for 48 h).

Elution is carried out with 1.5 L of toluene / ethyl acetate (70/30, v / v). The fractions containing the ester are collected and the solvent evaporated at 40 ° C under reduced pressure.

Preparation of assemblies with amylose The various solutions (chlorogenic acid, lipophilic derivatives of acylated and alkylated chlorogenic acid) are mixed with the amylose to determine their possible complexing activity towards the latter. The amylose / chlorogenic acid (or with hexadecyl chlorogenate or C16 acyl derivatives of chlorogenic acid) assemblies were made from 1% amylose gels. The construction protocol of the assemblies is initially adapted from that of Biais et al., 2006 (Carbohydrate Polymers 66 (3): 306-315). Five milliliters of a solution of chlorogenic acid (or lipophilic derivatives of chlorogenic acid) are prepared as follows: 10 mg of "molecule of interest" (chlorogenic acid, hexadecyl chlorogenate or C16 acyl derivatives) chlorogenic acid) are introduced into a volume of 5 ml of previously degassed water (15 min to avoid oxidation phenomena). This solution is then placed in an airtight bottle and heated at 90 ° C. for 30 minutes (in a bath of silicone oil) with magnetic stirring and then returned to ambient temperature.

Two hundred milligrams of amylose are introduced into an airtight bottle containing 20 ml of water previously degassed with nitrogen. This solution is heated at 160 ° C for 45 minutes with strong magnetic stirring and then cooled to 90 ° C. In parallel, 2.5 ml of the filtrate of the chlorogenic acid solution (or lipophilic derivatives of chlorogenic acid) are heated to 90 ° C.

When both solutions are at 90 ° C, the amylose solution is introduced into the vial containing the solution of chlorogenic acid (or lipophilic derivatives of chlorogenic acid). This solution is maintained at 90 ° C for 10 min with strong magnetic stirring, then cooled to room temperature and stored in the flask for 48 hours. The solution is centrifuged at 2000 g for 5 min; a portion of the precipitate is recovered, weighed in a tare box, and placed under a saturated NaCl atmosphere (water activity aw = 0.75 at ambient temperature) until mass equilibration (about 72h).

An amylose solution, without the addition of chlorogenic acid (or lipophilic derivatives of chlorogenic acid), is prepared under the same conditions so that the results obtained can be compared with a reference.

Characterization of Amylose / Chlorogenic Acid Complexes (or Lipophilic Derivatives of Chlorogenic Acid) and Reference Amylose Gel To characterize amylose / chlorogenic acid (or lipophilic derivatives of chlorogenic acid) assemblages obtained from the amylose / chlorogenic acid gel (or lipophilic derivatives of chlorogenic acid) after conditioning under NaCl. The starting molecules (chlorogenic acid, lipophilic derivatives of chlorogenic acid) are also studied.

The characterization of amylose / chlorogenic acid (or alkyl or acyl derivatives of chlorogenic acid) assemblies, and the reference amylose gel, required three different techniques, namely differential scanning calorimetry (DSC), diffraction of X-ray (XRD) and solid NMR.30 - X-ray diffractometry or "XRD" Twenty milligrams of sample, equilibrated at aw = 0.75, are placed in a copper washer between two sheets of adhesive tape, for prevent any change in moisture content.

The sample was examined by X-ray diffractometry or XRD. Measurements were made using a D8 Discover spectrometer with GADDS detector and crossed with Bruker-AXS mirrors, working at 40 kV and 40 mA, with a copper monochromator (X = 1.54059 A) and an alignment of the sample by microscopic video and laser.

The data was collected using a 120 ° curve detector for 10 minutes and normalized between 3 and 30 °. (20) - Differential scanning calorimetry Q100 (differential scanning calorimetry or DSC) The AED thermogram is recorded using a TA Instruments Q100 Scanning Differential Scanning Calorimetry Machine, and eight milligrams of sample and 12 μl of nitrogen-degassed water are introduced into a stainless steel cap (TA Instruments). The reference is a capsule containing 12 μl of water The capsules are then sealed with a heat-resistant seal and placed in the oven of the device The selected kinetics include a first cycle with an increase of 3 ° C / min of 1 C. to 140 ° C., followed by cooling from 3 ° C./min to 1 ° C. A second temperature cycle was carried out at 3 ° C./min up to 160 ° C. sample is rebalanced at room temperature at fast speed, without recording data. Solid-state NMR spectrometry This study was carried out on samples previously equilibrated in saturated NaCl saline solution.

The 130 solid-state NMR spectra (CP-MAS) were recorded on a 400 MHz Bruker Wide Bore spectrometer equipped with a double-resonance 4 mm H / cross-polarization-magic-angle-spinning (CP-MAS) probe. X. The CP-MAS sequence was performed with a 7 KHz rotation, a 90 ° proton pulse of 3.75 μs, a 7s recycle delay and a 30ms acquisition time during which dipole decoupling was applied. The contact time was 1 ms. The chemical shifts of the carbons were calibrated against the chemical shift of the glycine carbonyl group at 176.03 ppm (21 ° C). Results and discussion Thermal properties of amylose suspensions The complexing capacity with amylose of chlorogenic acid, and its lipophilic derivatives, is determined by differential scanning calorimetry, in comparison with ligand-free amylose. Amylose alone (without ligand) - reference The suspension of amylose alone (without ligand) was obtained following the same protocol as that used for the amylose / ligand suspensions. The thermogram obtained (Figure 1), during the differential analysis (DAL) study, serves as a reference for the rest of the study. During the first cycle, an endotherm at 108 ° C is observed. During the experiment, the amylose solution was heated to 145 ° C to solubilize the amylose, or the melting temperature of the amylose used was 148 ° C, which was verified by AED in excess water (thermogram not shown). At 145 ° C only part of the amylose is fused (the least well organized) and then retrograde rapidly on cooling.

The endotherm at 108 ° C corresponds to poorly retrograded amyloidosis with poor crystal organization. In the second cycle, two endotherms are observed: at 120 ° C and 145 ° C.

These two endotherms respectively correspond to the melting of the amylose retrograded during the descent in temperature carried out between the two cycles and the fusion of the initial amylose, having a good crystalline organization. - Chlorogenic acid and lipophilic derivatives of chlorogenic acid Differential enthalpic analysis was carried out on the ligands, in excess of water, in the temperature range from 20 ° C to 140 ° C with a kinetics of 3 ° C / min.

The thermograms obtained (not shown) show an endotherm, respectively, at: 65 ° C for chlorogenic acid, 51 ° C for 4-0-palmitoyl chlorogenic acid, 91 ° C for 3-0-palmitoyl acid chlorogenic, and 84 ° C for hexadecyl chlorogenate. - Assembly with amylose The thermograms obtained for the different assemblies are illustrated in Figures 2 to 5. The endotherms are identical for the four assemblies, with: - during the first cycle, an endotherm at 108 ° C, and - during the second cycle, an endotherm at 120 ° C and 148 ° C. These endotherms are similar to those of amylose alone (Figure 1). For the amylose / acyl derivative assembly of chlorogenic acid, an endotherm at 82 ° C is visible at the first and second cycles (Figures 4 and 5). This endotherm can not correspond to the lipophilic derivatives alone (melt temperature at 51 ° C and at 91 ° C for 4-0-chlorogenic palmitoyl acid and 3-0-chlorogenic palmitoyl acid, respectively). The fact that this endotherm is visible during the second cycle shows that this reaction is reversible. All indications are that this endotherm corresponds to the fusion of amylose / 4-0-palmitoyl chlorogenic complexes and amylose / chlorogenic 3-0-palmitoyl acid complexes. It should be noted, however, the low melting temperature (82 ° C) of these complexes. In the literature, the temperature of the amylose / lipid complexes is around 110 ° C. (for example, for palmitic acid, the melting temperature of the complexes is 112 ° C.). For the amylose / hexadecyl chlorogenate assembly, an endotherm around 83 ° C is also visible, but disappears in the second cycle (Figure 3). The melting temperature of this alkylated derivative being 84 ° C., the endotherm seems to correspond to the pure and uncomplexed alkyl derivative. No endotherms additional to those of amylose are apparent on the thermogram of amylose / chlorogenic acid assemblages (Figure 2).

In conclusion, the differential enthalpic analysis seems to conclude that there is no interaction between amylose and chlorogenic acid on the one hand, and amylose and the alkylated derivative (chlorogenate) on the other hand. hexadecyl). On the other hand, there are interactions between, on the one hand, amylose and chlorogenic 4-O-palmitoyl acid and, on the other hand, amylose and chlorogenic palmitoyl 3-O-acid. Crystalline Structure of In Vitro Assemblies An X-ray diffraction study was conducted on amylose alone (Figure 6A) and the four assemblages (amylose / chlorogenic acid - Figure 6B, amylose / hexadecyl chlorogenate - Figure 6E, amylose / 3-0- chlorogenic palmitoyl acid - Figure 6D and amylose / chlorogenic 4-0-palmitoyl acid - Figure 6C). All samples were previously placed under saturated NaCl atmosphere for one week.

The X-ray diffraction patterns obtained are shown in FIG. 6. The X-ray diffraction pattern obtained for amylose alone and used as a reference has a characteristic structure of type B (FIG. 6A). In this case the reflections obtained show that the amylose has demoted to double helices in a hexagonal stack. In the presence of chlorogenic acid or lipophilic derivatives of chlorogenic acid, X-ray diffraction also shows a type B structure (Figs. 6B-6E). This structure is explained by the presence of retrograded amylose due to the incomplete solubilization of the latter during the assembly formation protocol. Nevertheless, it is curious not to also observe characteristic V-type lines, i.e. characteristic of crystalline complexes formed with amylose for amylose / acyl derivative assemblies.

In conclusion, the reflections obtained are identical for all the assemblages and are characteristic of the crystalline type B which is explained by the presence of retrograded amylose. These results are in agreement with those obtained in AED for the amylose / chlorogenic acid and amylose / hexadecyl chlorogenate assemblages, but this technique does not allow us to conclude on the complexation of amylose and acylated derivatives of chlorogenic acid. . NMR of the solid The 130 CP / MAS NMR spectra obtained are shown in FIG. 7.

A type B spectrum is characterized by a doublet of lines of equal intensity around 100ppm. This multiplet is explained by considering that the repetitive unit that generates the double helix is maltose. However, all the spectra for amylose alone (FIG. 7A) or assemblages (amylose / chlorogenic acid - FIG. 7B, amylose / lipophilic derivatives of chlorogenic acid - FIGS. 7C to 7E) have a doublet at 100 ppm. This confirms the presence of amylose retrograded type B in all samples. For amylose / acyl derivatives of chlorogenic acid (chlorogenic 3-0-palmitoyl acid and 4-0-chlorogenic palmitoyl acid), an intense line at 102.6 ppm is still observed (FIGS. 7D and 7C). However, this line is characteristic of type V amylose spectra (single helix). This is in agreement with the uniqueness of the ct- (1-4) conformations in the meshes of simple helices. Moreover, for these two amylose / acylated derivatives of chlorogenic acid, the peak at 61 ppm has a large shoulder which corresponds to the presence of two types of left-left and left-trans orientations (FIGS. 7C and 7D).

For type B structures, the width of the peak is small, the left-trans orientation is predominantly predominant, the left-left orientation is almost non-existent and therefore undetected, whereas in the case of a structure of type V the two types of left-left and left-handed orientations are clearly expressed.

In the case of the two amylose / acylated derivatives of chlorogenic acid (Figures 7C and 7D), the presence of both crystalline types V and B explains the left-trans orientation as being predominant but not unique. We are therefore in this case in the presence of simple propellers (type V) and double helices with a crystalline arrangement of type B.

In conclusion, this study allows us to affirm the presence of simple helices in the case of acylated derivatives of chlorogenic acid complexed with amylose, and to confirm the absence of interaction between, on the one hand, the amylose and chlorogenic acid and, on the other hand, amylose and hexadecyl chlorogenate. General Conclusion In the present study, the different assemblies, and their respective signatures of the calorimetric, X-ray and solid-state NMR type, were observed and compared to the reference constituted by ligand-free amylose. The techniques used show excellent complementarity in the determination of important structural features such as crystalline type, helical conformation or the nature of inclusion. The X-ray diffraction data only show type B structure spectra, whatever the assemblages formed with amylose (chlorogenic acid, acylated derivatives or alkyls); these results do not make it possible to determine the nature of the possible interactions, in particular the presence of simple helices characteristic of the formation of clathrates. For the amylose / chlorogenic acid and amylose / alkyl derivative assemblages, the results of differential enthalpy analysis and solid MNR confirm the absence of complexes.

For the amylose / acyl derivative assemblies, the presence of the complexes is shown, on the one hand, by the presence of their fusion endotherms in differential enthalpy analysis and by the reversibility of their formation during cooling and on the other hand by the presence of an intense singlet at 102.6 ppm in solid NMR.

These data allow the demonstration of the presence of simple propellers and to affirm the absence of an inter-helix space allowing the capture of bulky molecules. These results, associated with the X-ray diffraction, make it possible to conclude that the complexes obtained are amorphous and / or structured inclusion complexes at a very small scale (low melting temperature, no X-ray diffraction signature), and that only the graft (aliphatic chain) is captured in the helical cavity of amyloidosis. This work has highlighted the difficulty of trapping, by amyloidosis, an imposing molecule such as chlorogenic acid and thus to protect it from oxidation. The synthesis of lipophilic derivatives of C16 chlorogenic acid by acylation, has shown that amylose could trap the grafted fatty chain by trapping in its helical cavity.

This study also showed the importance of the position of the aliphatic chain grafted onto the ring, with regard to the better complexation of chlorogenic 4-O-palmitoyl acid with respect to chlorogenic 3-O-palmitoyl acid. The present results show in particular that chlorogenic 4-O-palmitoyl acid forms an amorphous complex with amylose, with only the grafted side chain included in the amylose cavity. These results make it possible to deduce the complex schematic model as illustrated in FIG. 8. Without being limited by any theory, the grafting of a longer aliphatic chain for the alkyl derivative, for example C18 or C20, should make it possible to to obtain a sufficient complexing effect with amylose, for the formation of a complex (the steric hindrance being the only factor limiting the complexation of the grafted chain by alkylation). Moreover, a major disadvantage in the addition of protective assemblies in the aqueous phase of an emulsion is the phase shift of the aqueous phase due to the nucleation and precipitation of the complex crystals. However, in our case, amorphous and / or structured inclusion complexes are obtained on a very small scale, the steric hindrance of the molecule not allowing a greater crystalline arrangement. Inclusion complexes are taken in the disorganized network of uncomplexed amylose chains, which should allow a clear reduction in the risk of precipitation in the aqueous phase. This work makes it possible to envisage in particular the protection of molecules that are sensitive to oxidation when they are incorporated in an emulsion or simply in an aqueous phase or an aqueous medium. The other interesting point of this work is the fact that the amylose / C16 fatty acid (palmitic acid) complexes are well known in the literature for their high stability against acid and enzymatic hydrolysis, suggesting that during the release of Chlorogenic acid, the saturated fatty acid will not be released, but will remain prisoner of amyloidosis until the end of digestion.

Claims (1)

1. A process for the preparation of complexes comprising amylose assembled with at least one organic molecule of interest, which process is characterized in that it comprises the following steps: (a) a lipophilization stage consisting in grafting a fatty aliphatic chain onto said organic molecule of interest, for the preparation of a lipophilic derivative of said organic molecule of interest, (b) a step of bringing into contact between (i) said lipophilic derivative of said organic molecule of interest bearing said grafted fatty aliphatic chain, resulting from said lipophilization step (a), and (ii) said amylose, and (c) a step of forming said amylose / lipophilic derivative complexes of the organic molecule of interest. 2. A process according to claim 1, characterized in that the organic molecule of interest is chosen from organic molecules having an antioxidant activity. 3. A process according to any one of claims 1 or 2, characterized in that the organic molecule of interest is selected from polyphenols. 4. A process according to claim 3, characterized in that the organic molecule of interest is chosen from phenolic acids. 5. A process according to claim 4, characterized in that the organic molecule of interest is chosen from hydroxycinnamic acids or esters of hydroxycinnamic acids. 6. A process according to claim 5, characterized in that the organic molecule of interest in the form of hydroxycinnamic acid is selected from ferulic acids, p-coumaric acids, ocoumaric acids, caffeic acids and sinapic acids. 7. A process according to claim 5, characterized in that the organic molecule of interest in the form of hydroxycinnamic acid ester is chosen from among caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and sinapoylquinic acids, or among rosmarinic acids. 8. A process according to claim 7, characterized in that the lipophilization step (a) comprises grafting the fatty aliphatic chain on the quinic group of the organic molecule of interest. 9. A process according to claim 8, characterized in that the lipophilization step (a) comprises grafting the fatty aliphatic chain on one of the hydroxyl functions of the quinic group of the organic molecule of interest. 10. A process according to claim 9, characterized in that the organic molecule of interest consists of 5-O-caféoylquinique, and in that the lipophilization step (a) consists in grafting the fatty aliphatic chain on the grouping. hydroxyl at position 3 or in position 4 of the quinic group of said organic molecule of interest. 11. A process according to any one of claims 1 to 10, characterized in that the lipophilic derivative of the organic molecule of interest, derived from the lipophilization step (a), comprises a fatty aliphatic grafted chain comprising a grouping linear alkyl. 12. A process according to claim 11, characterized in that the grafted fatty aliphatic chain comprises a chain length of between 12 and 16 carbon atoms, inclusive. 13. A process according to any one of claims 1 to 12, characterized in that the step of forming the complexes (c) is carried out so as to obtain inclusion complexes, in which the aliphatic chain greasts Lipophilic derivatives of organic compounds of interest are included in one of the helices of the associated amyloidosis. 14. A process according to any one of claims 1 to 13, characterized in that, during the contacting step (b), the amylose molecule consists of amylose alone or of amylose in the form of starch. 2 9812 74 47 15. Organic chemical compound in the form of a complex, comprising amylose assembled with at least one organic molecule of interest, characterized in that said organic molecule of interest is in a lipophilic derivative form carrying a chain aliphatic 5 fat grafted. 16. Organic chemical compound according to claim 15, characterized in that the organic molecule of interest bearing the grafted fatty aliphatic chain, is selected from organic molecules having an antioxidant activity. 10 17. Organic chemical compound according to any one of claims 15 or 16, characterized in that the organic molecule of interest is selected from polyphenols. 18. Organic chemical compound according to claim 17, characterized in that the organic molecule of interest is chosen from phenolic acids. 19. Organic chemical compound according to claim 18, characterized in that the organic molecule of interest is chosen from hydroxycinnamic acids or hydroxycinnamic acid esters. 20. An organic chemical compound according to claim 19, characterized in that the organic molecule of interest in the form of hydroxycinnamic acid is selected from ferulic acids, coumaric acids, o-coumaric acids, caffeic acids and sinapic acids. 21. An organic chemical compound according to claim 19, characterized in that the organic molecule of interest in the form of hydroxycinnamic acid ester is chosen from among caffeoylquinic acids, p-coumaroylquinic acids, feruloylquinic acids and acids. sinapoylquiniques, or among the rosmarinic acids. 22. An organic chemical compound according to claim 21, characterized in that the lipophilic derivative of the organic molecule of interest carries the fatty aliphatic chain grafted onto its quinic group. 23. The organic chemical compound as claimed in claim 22, characterized in that the fatty aliphatic grafted chain is carried by one of the oxygens covalently bonded to the cyclohexyl group of the quinic group. 24. The organic chemical compound as claimed in claim 23, characterized in that the lipophilic derivative of the organic molecule of interest consists of a 5-O-caféoylquinique, whose grafted fatty aliphatic chain is carried by the oxygen atom. position 3 or in position 4 of the quinic group. 25. An organic chemical compound according to any one of claims 15 to 24, characterized in that the grafted fatty aliphatic chain comprises a linear alkyl group. 26. The organic chemical compound as claimed in claim 25, characterized in that the grafted fatty aliphatic chain comprises a chain length of between 12 and 16 carbon atoms, inclusive. An organic chemical compound according to any of claims 15 to 26, characterized in that said organic chemical compound is in the form of an inclusion complex, wherein the fatty aliphatic chain grafted lipophilic derivatives of organic compounds of interest is included in one of the helices of the associated amyloidosis. 28.- Composition incorporating the organic chemical compound according to any one of claims 15 to 27.