EP2776472A1

EP2776472A1 - Method for preparing glucans from aspergillus niger

Info

Publication number: EP2776472A1
Application number: EP12772928.3A
Authority: EP
Inventors: Philippe Richard VAESEN; Olivier Yvette Marcel BAUM
Original assignee: Kitozyme SA
Current assignee: Kitozyme SA
Priority date: 2011-10-11
Filing date: 2012-10-11
Publication date: 2014-09-17
Also published as: FR2981075A1; WO2013053838A1; CA2851576A1; US20140220078A1

Abstract

The present application relates to a method for preparing glucans from Aspergillus niger, characterized in that it includes: (i) at least partially deacetylating the mycelium of the Aspergillus niger; (ii) treating the deacetylated or partially deacetylated mycelium with acid, preferably after purification and/or washing, in order to obtain insoluble glucans and soluble chitosan, wherein said acid treatment includes contacting the deacetylated mycelium with an acid solution; (iii) separating the soluble chitosan from the glucans; (iv) treating the glucans with an alkali, which includes contacting the glucans with an alkali solution so as flocculate the glucans; and (v) drying the flocculated glucans in order to obtain a glucan powder. The present invention further relates to the resulting glucans, to compositions including said glucans, and to the uses thereof. The glucans of the invention can be used as immunostimulants.

Description

Process for the preparation of glucans from Aspergillus niger

The present invention relates to a process for preparing glucans from Aspergillus Niger, the Beta-glucans thus obtained, and their uses.

It has been known for a long time to prepare beta-glucans from various substrates of natural origin, for example beta (1, 4) glucans derived from cereals, beta (1, 3) (1, 6) glucans derived from yeast and mushrooms. In particular, it is particularly known to prepare glucans from yeast, as typically Saccharomyces cerevisiae. EP 0759 089 discloses that glucans derived from Saccharomyces cerevisiae provide various beneficial effects to fish and other animals. Various chemical or biochemical treatments are carried out to eliminate beta- (1-6) -linked glucan chains, in particular by treatment with beta- (1 -6) -glucanase. These treatments are envisaged to improve the immunostimulatory activity of glucans by increasing the proportion of beta-linked glucans (1-3), which would be related to the aforementioned activity. However, the literature evokes strong structural divergences according to the origin of glucans. Novak (Novak et al., Development of Views on Beta-Glucan Composition and Structure, Biology and Chemistry of Beta Glucans, Vol 01, 201 1, 1-9) can typically be referred to. This article teaches that glucan preparations are heterogeneous depending on the organism used to extract them. Because of this heterogeneity, it is difficult to know exactly the structure of glucans and to draw any hasty conclusions about their properties, especially biological. In particular the degree of branching of lateral links on the beta-glucan skeleton depends on the genus, the species, and the strain of the organism considered. However, the degree of branching is important for the macromolecular structure of glucans which is linked to immunostimulatory properties.

Aspergillus niger is known primarily as a source of chitin to produce chitosan. Aspergillus niger mycelium is a by-product of A. niger fermentation for citric acid production. Methods of obtaining chitosan from A. niger are for example described in the international application KitoZyme WO03068824. Glucans are an insoluble by-product obtained during the preparation of chitosan. The glucans resulting from the chitosan preparation are discarded. It can be seen from the teaching of international applications WO0168714 and WO03086281, which do not envisage the subsequent treatment of insoluble glucans.

A priori there is no specific study on glucans obtained from Aspergillus niger. However, as described above, the complex structure of the glucans does not make it possible to advance on the immunostimulatory properties of the original glucans. of Aspergillus niger. Any conclusion in this sense would be pure speculation.

On the other hand, if glucans can be obtained as an insoluble by-product during the preparation of chitosan, the methods known to date do not allow industrial exploitation of glucans coproduced with chitosan obtained from chitin. In addition, the degree of purity of glucans is generally not satisfactory. The chitosan preparation process of the international application WO03068824 of KitoZyme does not make it possible to obtain glucans with satisfactory purity since the glucans are in the form of chitin-glucan copolymers. This international application indicates that a chitin-glucan copolymer may possess from 30 to 50% (m / m) of chitin and between 50 and 70% of beta-glucans. However, it is a copolymer having bonds between chitin and glucans. This product glucans bound to chitin in the form of copolymer. For chitosan production from chitin, chitin is converted using an alkaline solution under "aggressive" conditions to deacetylate chitin. However, these conditions suggest that the glucans obtained as a by-product will have a sufficiently deteriorated structure so that it is not envisaged to use them as immunostimulatory glucans, which explains why the prior art excludes this fraction.

In this application:

By "Aspergillus niger glucans" or "Aspergillus niger glucans", or "Aspergillus niger originated glucans", or "Aspergillus niger glucans", are meant beta-glucans obtained or susceptible to be obtained from the organism Aspergillus niger, and in particular from mycelium Aspergillus niger. This name can be transposed to other products than glucans, such as chitosan or chitin.

On the other hand, reference is made to "glucans" according to the present invention, but this designation relates more specifically to an extract of Aspergillus niger comprising predominantly in bulk glucans, the remainder of the extract being constituted by impurities in the sense of the invention, that is to say compounds other than glucans.

The terms "glucans" or "beta-glucans" are used here as synonyms. By "immunostimulatory glucans" is meant glucans having properties promoting the immunostimulation of at least one reference marker of the immune system, such as, for example, the neutrophils of the innate immune system.

The present invention aims to provide an industrial process for the preparation of glucans from Aspergillus niger.

The present invention in particular aims to improve the yield of a process for preparing glucans from Aspergillus niger. The present invention in particular to improve the yield of a process for preparing glucans from Aspergillus niger with the co-production of chitosan. The present invention also aims to purify glucans from Aspergillus niger according to an industrial process.

Another object of the present invention is to obtain or prepare immunostimulatory glucans, in particular for the stimulation of the immune system of an animal or of a human being, especially by oral nutrition, application to the skin, etc.

It has surprisingly been found that the above objects can be satisfied by the present invention.

In particular, the invention relates to a method or method for preparing glucan from Aspergillus niger mycelium, said method comprising: (i) at least partial deacetylation of Aspergillus niger mycelium; (ii) acid treatment of the deacetylated or partially deacetylated mycelium, preferably after purification and / or washing, to obtain insoluble glucan and soluble chitosan, said acidic treatment comprising contacting the deacetylated mycelium with an acidic solution; (iii) the separation of soluble chitosan on the one hand, and insoluble glucan on the other; (iv) an alkaline treatment of glucan comprising contacting glucan with an alkaline solution to flocculate glucan; and (v) drying the flocculated glucan to obtain a glucan powder.

The glucan of the invention is never 100% pure, but includes secondary substances. Among the secondary substances it is possible to note the presence of ash, lipids, chitin and / or chitosan or other polysaccharides (for example mannan), which one seeks to minimize, with a view to improving the purity thereof. with the intended use. By "deacetylation of chitin" is meant the total or partial deacetylation of chitin because chitosan may comprise a degree of acetylation more or less high.

Step (i) deacetylation

Advantageously, the deacetylation (step (i)) is carried out by bringing the mycelium into contact with an alkaline material, preferably bringing hydroxide ions, and preferably with sodium hydroxide (NaOH), at a concentration of a temperature, and for a time sufficient to deacetylate the chitin to chitosan with preferably a minimum yield of 4% to chitosan, and preferably greater than 9% and obtain a chitosan preferably having a degree of acetylation, that is, that is to say a molar proportion of N-acetyl-D-glucosamine units along the chitosan chains, from 0 to 50%, and preferably from 10 to 25%. In a preferred embodiment, the alkaline solution comprises a concentration greater than 40% (mass / volume) of alkaline material providing hydroxyl ions relative to the total mass of the alkaline solution. By yield is meant the mass ratio expressed in% of the dry mass of the fraction containing chitosan (chitosan and its impurities) on the dry mass of the starting mycelium. For example, a solution of sodium hydroxide of concentration 50% (mass / volume) is used.

According to one variant, the Aspergillus niger esX mycelium is mixed with a concentrated alkaline solution. Preferably, the alkaline solution is chosen from an aqueous solution of sodium or potassium hydroxide, carbonate or sodium bicarbonate. Advantageously, the ratio by mass of the alkaline material with respect to chitin is between 0.1 to 5, and preferably between 0.3 to 3, and more preferably from 0.5 to 1.5 (m / m). .

Preferably the treatment with the alkaline solution of the Aspergillus niger mycelium is carried out at a temperature of between 50 and 1 × 20, and more preferably between 80 and 1 ° C.

Firstly, the mycelium can be brought into contact with the alkaline solution, then a gradual rise in temperature can be achieved. For example the temperature reaches 110 ° C in 2 hours, then the temperature is maintained for 6 hours, then the resulting suspension is transferred to the next step in 1 hour, the temperature decreases to room temperature.

According to another preferred embodiment, the deacetylation step itself is carried out for a period of between 30 minutes and 10 hours, and preferably between 3 and 8 hours.

It is preferable to perform a thorough deacetylation in temperature and reaction time to increase the yield of chitosan (according to Table 3 of the examples). For example, deacetylation can be carried out by bringing the mycelium into contact with the alkaline solution for a period of between 4 and 7 hours, then increasing the temperature with maintenance at a temperature of between 50 and 120 ° C., and even more preferably between 80 and 120 ° C.

The insoluble fraction in an alkaline medium can be separated from the soluble fraction by filtration and / or centrifugation.

The insoluble alkali fraction obtained after deacetylation is preferably suspended, then diluted, filtered and washed with water. This or these steps are typically performed as necessary.

Preferably the alkaline solution is recovered after this first step, concentrated, recycled and reused for the deacetylation of chitin. Prior to the deacetylation step, the method of the invention may comprise the pretreatment of Aspergillus niger mycelium. The mycelium is preferably washed, and concentrated and / or dried. This allows according to one variant to use a mycelium completely or partially discolored. The white or beige color may be advantageous for applications such as pharmacy, nutraceutical, cosmetic, or in the food industry. Filtered / washed mycelium advantageously provides an improvement in the yield of chitosan and / or glucan.

Stage (ii) acid treatment

This step is carried out under conditions making it possible to obtain a soluble fraction and an insoluble fraction. The insoluble fraction in an alkaline medium is brought into contact with an acid solution according to step (ii), the pH being advantageously adjusted to a value of less than 6.5, and preferably less than 5.5, by adding an acid such as for example chosen from hydrochloric acid, lactic acid, formic acid, glutamic acid, phthalic acid, succinic acid, glycolic acid, citric acid and, preferably, acetic acid. The reaction may for example be carried out at room temperature or any other temperature favoring the separation of the soluble fraction containing chitosan from the insoluble fraction containing glucan.

An acidic solution having a concentration of between 0.1 and 1 N can be used as an acidic solution. For example, an 80% solution of acetic acid is used.

Advantageously, the acid treatment of step (ii) comprises the addition of an organic acid until a pH of between 3 and 5.5 is obtained, and preferably between 3.5 and 4.5. .

Preferably, the acid treatment of step (ii) comprises or consists of the addition of acetic acid to the deacetylated mycelium obtained in step (i), optionally washed, dried and / or concentrated.

Preferably, prior to step (ii) the alkaline residue is removed by several washes with water.

Step (iii) separation of glucan and chitosan

Preferably, the separation according to step (iii) is carried out by separation using a continuous nozzle centrifuge. More particularly, for this centrifugation step can be used a nozzle separator type centrifuge or self-debirking type (eg NA7), or a high performance BTUX vortex separator type centrifuge (both). types of centrifuge NA7 or BTUX being marketed respectively by Westfalia and AlfaLaval).

According to one variant, the method of the invention may comprise an additional step (iiia) for treating the soluble chitosan obtained in step (iii) to separate the insoluble glucan, this additional step possibly being repeated one or more times. The insoluble glucan obtained according to this additional step (iiia) may be added to the insoluble glucan recovered in step (iii).

According to a preferred variant, step (iiia) implements a decanter with centrifugal force (for example a Sedicanteur marketed by Flottweg, which compared to a conventional clarifier has a biconical bowl and a higher centrifugal speed than a conventional decanter ). Such a decanter comprises a solid bowl centrifuge and a conveyor screw. It can typically develop a centrifugal force of 6,000 to 10,000g. The speed of the bowl, the speed of the screw and the layer height are set by the skilled person to optimize the performance of the machine.

Step (iv) separation of chitosan and "flocculation packaging" of glucan

The glucan according to the invention is insoluble in water at room temperature and at acid pH, less than 7. Chitosan is soluble under these conditions. The insolubility of glucans makes it possible to purify them. The flocculation step advantageously makes it possible to separate the glucans by industrial means and in a time allowing a profitable industrial exploitation.

Surprisingly, it has been discovered in the present invention that simple treatment with sodium hydroxide solution prior to drying does not permit satisfactory liquid separation. In particular, it has been found that the product obtained by contacting the insoluble extract obtained according to step (iii) is not mechanically strong enough to achieve a satisfactory solid / liquid separation. The product is denatured and can not recover a satisfactory percentage of glucan in the final extract. By carrying out the flocculation step according to the present invention, the product is made more mechanically resistant to a solid-liquid separation step. At least partially glucans can be dehydrated, water removed more easily, and thus recovering final extract with lower ash content and thus higher glucan content.

Preferably the alkaline solution used in the flocculation step (iv) comprises or consists of lime, for example in the form of 30% lime milk (Ca (OH) ₂ ) (calcium hydroxide), which precipitates in a solid easily recoverable and resists the mechanical constraints of conditioning by allowing recovery of glucans with a degree of purity in fine satisfactory.

Alternatively, the alkaline solution of flocculation step (iv) comprises or consists of a mixture of sodium hydroxide (NaOH) and milk of lime (Ca (OH) ₂ ). When such a sodium hydroxide / lime (calcium hydroxide) mixture is used, a weight ratio of sodium hydroxide / lime (calcium hydroxide) of 1/2 to 1/10, and preferably of about 2 to 1/6 (preferably about 1 / 3.5).

This step is preferably followed or controlled by pHmetry. It is preferred to stop the addition of alkaline solution when the pH becomes greater than 9 and preferably greater than 10.

Flocculation of glucan is important to allow separation easily exploitable industrially. It is preferred to use a sodium hydroxide / calcium hydroxide mixture in order to limit calcium ion intake in glucans to avoid generating excess ash. The presence of lime advantageously confers a glucans texture facilitating their separation in an industrial equipment. This also allows a reduction in separation times which is industrially very interesting.

It is important to perform the flocculation step to improve the productivity and purity of the resulting glucans. This flocculation step is also important to allow drying of glucan (via a dryer type "flash dryer") in the form of powder.

Generally after the flocculation step the glucan is in the form of an alkaline suspension (pH greater than 10). Step (iva) additional purification of glucans

The glucan obtained in step (iv) may optionally be re-purified before the drying step, in order to adapt the purity of the glucan to the intended use, in particular to increase the purity, for example by decreasing the residual ash content in glucan. This additional step comprises bringing the glucans into contact with a solution for solubilizing chitosan and then removing the solubilized chitosan by separation.

This additional purification step is advantageous if it is desired to increase the proportion by mass of glucan beyond 80% relative to the total mass of the final product.

The additional purification step preferably comprises contacting the flocculated glucans with an acidic solution. Preferably, the acid solution in question has a pH of greater than 5 and less than 7, and even more preferably between 6 and 6.6. Acetic acid is preferably used to prepare this acidic solution.

A separation can be carried out with a filter press or decanter, basket centrifuge, high centrifugal force decanter (Sedicanteur®), or band filter.

The glucan obtained according to step (v) can then be dried.

Stage (v) of glucan drying

This step is advantageously carried out by introducing the glucan obtained in the previous step (step (iv) or (iva)) into a drying device to remove the residual liquids.

These devices are known to those skilled in the art.

Advantageously, it is possible to use a glucan drying ("flash dryer"), in order to obtain a fine powder of beige to slightly brown color.

This drying may optionally be followed by granulation. The final product is then packaged.

Between each step described above, i.e. between steps (i) and (ii), (ii) and (iii), (iii) and (iv), and / or (iv) and v), it is possible to perform different treatments. These treatments may for example consist of one or more washes, purifications, concentrations, separations, and / or one or more other treatments intended to improve the yield and / or purification of the products obtained according to the process of the invention.

According to one variant, the process of the invention does not comprise a glucan oxidation step.

The method of the present invention makes it possible to improve the yield of chitosan by transforming a maximum amount of chitin into chitosan ("deacetylation") while recovering glucan satisfactorily, and preferably so as to be able to valorize them as an immunostimulatory agent.

In another aspect, the present invention relates to a method for co-preparation of chitosan and glucan from Aspergillus niger, said method comprising the glucan preparation according to the method of preparation described above, including any variant and any embodiment particular, possibly combined with each other, and the preparation of chitosan.

In another aspect, the invention relates to a method for the production of chitosan and glucans from chitin of Aspergillus niger origin comprising the following steps: (a) bringing chitin into contact with a basic solution for deacetylating at least partially chitin and recover a soluble fraction in an alkaline medium and an insoluble fraction in an alkaline medium, (b) contacting the insoluble fraction in an alkaline medium with an acidic solution, to obtain a soluble fraction of chitosan, more or less acetylated, and an insoluble fraction comprising glucans and then (c) further treating chitosan to wash, purify, and optionally dry it, said method also comprising treating the glucans obtained in step (b) to wash, purify, and optionally dry them.

The different steps (i) to (v), including their variants apply to this aspect as well.

The alkali-soluble fraction obtained after step (a) is generally discarded because it contains different salts, proteins, and hydrolysed soluble glucans.

Alternatively, the soluble chitosan obtained in step (b) may be contacted with a chitin deacetylase enzyme to obtain chitosan.

The prepared chitosan may be of high molecular weight. The chitosan may also be of low or medium average molecular weight.

By low average molecular weight is meant average molecular weight less than 100,000. By high average molecular weight is meant a chitosan having an average molecular mass greater than 100,000. Preferably, the chitosan is of very low average molecular weight, that is to say less than 50,000. Preferably, chitosan has an average molecular weight of between 10,000 and 50,000. The average molecular weights are determined by measurement with a Ubbelohde type capillary viscometer or by steric exclusion chromatography with light scattering detection (SEC-MALLS) or by triple detection (for example with the Viscotek Triple Detector Array Max System). It is possible to hydrolyze chitosan to reduce its molecular weight.

Advantageously, chitosan is obtained by controlling the degree of acetylation.

By the term "chitosan having a controlled degree of acetylation" is meant a product whose degree of acetylation, i.e. the proportion of N-acetyl-glucosamine units can be adjusted in a controlled manner.

For the treatment of chitosan, one can refer in particular to the conditions described in the application WO03068824 KitoZyme.

It is also interesting to note that, according to a preferred embodiment, the process of the invention for extracting glucan and chitosan from the Aspergillus niger mycelium lasts less than 12 hours, and preferably less than 10 hours. .

Beta-qlucan According to another aspect, the invention relates to beta-glucan derived from Aspergillus niger obtainable by a method as defined according to the invention, including any variant and any particular embodiment, possibly combined with each other.

According to one variant, the glucan of the invention is suitable for animal feed, in particular that its composition is suitable for animal feed. In particular, glucan has a glucan content greater than 50%, preferably greater than 55%, and more preferably greater than 60%, especially for animal feed.

According to another variant, the glucan of the invention is suitable for application in humans, in particular for oral administration or application to the skin, and in particular food grade, cosmetic and / or pharmaceutical. Preferably, the glucan has a purity greater than 70%, preferably greater than 75%, and more preferably greater than 80%, especially for applications in humans. These percentages are expressed as weight of glucan relative to the total mass of wet product. By wet product is meant the finished product with its residual water content, which is in a preferred variant of 4 to 10%.

It has been found after the analysis of the glucan obtained that it has predominantly glycosidic linkages between beta-type glucose units (1, 3). In particular, it has been very surprisingly observed that the process of the invention preserves the bioactivity of the glucan obtained, that is to say its ability to modulate the immune system. As mentioned above, the literature shows that the activity of beta (1, 3) -glucan strongly depends on its structure (glycosidic bonds, branching, etc.), which can be affected by the treatments it undergoes. Contrary to what one could think, in spite of the very hydrolysing conditions used for the deacetylation of chitin in chitosan, the glucan obtained according to the process of the invention has an activity of modulation of the immune system, as demonstrated by oral administration in the fish Pimephales promelas, whose parameters of the innate immune system are studied.

Preferably, the glucan obtained has a high proportion of beta- (1 -3) glycoside bonds, for example greater than 70% relative to the total number of glucan bonds. It is known that the higher the proportion of beta- (1 -3) bonds, the better the immunostimulatory properties.

This glucan is advantageously obtained without the use of a beta-glucanase enzyme.

Preferably, the glucan of the invention comprises less than 15% by weight of chitosan with respect to the total mass of wet product. Such glucans are satisfactory for oral administration in animals. According to one embodiment, the glucan comprises less than 10% by weight of chitosan relative to the mass of wet product. These glucans are suitable for administration in humans, oral or on the skin.

The percentage by mass of chitosan present in glucan can be analyzed according to the chitosan extraction method by precipitation / washing / weighing.

Preferably, the glucan of the invention comprises less than 10% of ash. It is also possible to obtain 5% of ash, and possibly less than 3% of ash with respect to the mass of wet product.

Preferably, the glucan of the invention comprises less than 5% lipid, and less than 1% protein relative to the wet moist mass.

Preferably, the glucan of the invention does not comprise detectable chitin. One can seek to detect the presence of chitin by the method of assaying sugars after hydrolysis and derivatization ("derivatization" in English), as described in the examples. Immunomodulating properties

The invention also relates to beta-glucan from Aspergillus niger for the modulation of the immune system of a human being or an animal.

An animal to which the glucan of the invention may be administered is typically selected from commercial animals, racing animals (horses, dogs), pets, and aquaculture fish.

According to one variant, glucan is intended for improving the functions of the immune system, for example neutrophils with regard to oral administration, and Langerhans cells with regard to application to the skin.

According to another variant, glucan is intended to increase the degranulation capacity and the activity of neutrophils.

The invention also covers a dietary supplement composition for humans or animals, comprising glucan from Aspergillus niger as defined above including any variant and any particular embodiment, possibly combined with each other.

The present invention also covers a solid food for fish, characterized in that it comprises glucan from Aspergillus niger as defined above. Typically, such food comprises a starch-providing substance, a protein-providing substance, a lipid-providing substance, optionally a mixture of antibiotics, and optionally a mixture of minerals and vitamins, optionally antioxidant substances, optionally digestive enzymes, possibly microorganisms with probiotic activity, possibly fibers with prebiotic activity. The starch-providing substances are generally derived from cereals such as, for example, soybean, corn, wheat, oats, or barley. Examples of protein-providing substances are fish meal, soybean meal, dehydrated whey, soy protein, soy flour, blood meal, plasma protein, dehydrated skim milk, concentrate of whey protein, rapeseed meal, corn gluten meal, wheat gluten meal, yeast, sunflower meal. The lipid-providing substances are generally selected from soybean oil, lecithin, coconut oil, whey lipids, lard oil, or sheep tallow.

The present invention further encompasses a pharmaceutical composition comprising glucan from Aspergillus niger as defined above.

The invention also covers a pharmaceutical composition comprising glucan from Aspergillus Niger as defined above.

The invention also covers a cosmetic composition comprising glucan from Aspergillus niger \ e \ as defined above.

According to another aspect, the invention relates to the use of glucans for applications in the medical, pharmaceutical, nutraceutical, cosmetic, agricultural, food, textile, and / or environmental fields. General use of chitosan and glucan - Formulations

The chitosan obtained according to the process of the invention can be used in various applications, and typically as those described in the application WO03068824 of KitoZyme.

The chitosan and glucan obtained according to the process of the invention can be used in the form of micro-, milli- or nanoparticles, which can be prepared by techniques known to those skilled in the art (for example: Polymeric Biomaterials, S Dimitriu ED, Marcel Dekker, 2002, Chap 1).

The glucan of the present invention is essentially insoluble directly in any solvent at 25 ° C at atmospheric pressure. Glucan can be prepared as a powder or lyophilized fiber.

The glucan of the invention may be mixed with one or more active substances, and one or more excipients. Thus, the invention also covers a formulation comprising the glucan of the invention.

The glucan of the invention can be prepared in the form of capsules, tablets, powder, gel, film, emulsion, or suspension. Glucan can also be included in delivery systems, such as vectors for example to be delivered at the level of the small intestine. Glucan can be included in food matrices such as bars, beverages, baked goods, or dairy products.

Alternatively, the glucan of the invention may be formulated into a topically applicable composition. It may in particular be cosmetic or pharmaceutical compositions applicable topically (applied to a keratin material, for example the skin). Such compositions may comprise cosmetically or pharmaceutically active ingredients (active ingredients) and / or topically acceptable excipients, known to those skilled in the cosmetics or pharmacy field. Specific examples of formulations according to the invention comprising glucan are oil-in-water and water-in-oil emulsions. The invention covers lipsticks and lip balms containing glucan.

For a better formulation in compositions applicable to the skin and to allow a pleasant feeling of a homogeneous composition and without solid particles detectable with the naked eye or by the skin, the glucan according to the present invention is preferably micronized, it is that is, reduced size of the glucan or glucan-containing particles to a size less than one millimeter and preferably less than 500 microns (μηι). It is advantageous that the diameter of 70%, and preferably 90%, of the particles is less than 350 microns (0.7) <355 μm, preferably d (0.9) <355 μm), preferably less than 100 microns, in particular for formulations of the oil-in-water and water-in-oil emulsion type, and preferably less than 50 microns for lipsticks and lip balm. The particle size is that obtained by laser diffractometry.

Alternatively, the glucan can be used after sieving.

For example, a fish feed composition is formulated as a gel comprising a premix of powder with 50% protein and vitamin C mixed with hot water (ΘΟ 'Ό) in a 1: 1 ratio to which the compounds of the invention are added, for example at a concentration of 0.1 to 5 g / kg of food. The gel formed after mixing is typically granulated (0.1 -1 mm) just before use to feed the fish.

In the figures:

FIG. 1 represents a schematic diagram of a variant of the method of the invention comprising steps (i) to (v);

FIG. 2 is a schematic diagram of another variant of the process of the invention comprising steps (i) to (v), and additional purification of the glucans according to step (iva); FIG. 3 represents a histogram of degranulation of the primary granules of neutrophils after supplementation of the fish with beta-glucans L1 1 (5g / kg of food), L15 (4g / kg) and beta-glucans derived from yeast (glucan = L04, 5g / kg) for 8 days, without stressing the fish;

FIG. 4 represents a histogram of oxidative burst index after supplementation of fish with beta-glucans L1 1 (5 g / kg of food), L15 (4 g / kg) and beta-glucans derived from yeast (glucan = L04, 5g / kg) for 8 days, with stress applied to fish after day 7;

FIG. 5 represents the evolution of the CD54 marker in a monocyte population as a function of the concentration of beta-glucan in the medium;

FIG. 6 shows the evolution of the CD54 marker in a population of plasmacytoid dendritic cells as a function of the beta-glucan concentration in the medium;

FIG. 7 represents the production of Interleukin 6 (IL-6) as a function of the beta-glucan concentration;

FIG. 8 represents the production of Tumor Necrosis factor alpha (TNF-α) as a function of the concentration of beta-glucan;

FIG. 9 represents the production of Macrophage Inflammatory Protein 1 alpha (MIP-1 oc) as a function of the concentration of beta-glucan.

Other objects, features and advantages of the invention will become apparent to those skilled in the art following the reading of the explanatory description which refers to examples which are given by way of illustration only and which can not in in no way limit the scope of the invention.

The examples are an integral part of the present invention and any features appearing novel from any prior art from the description taken as a whole, including the examples, form an integral part of the invention in its function and its generality.

Thus, each example has a general scope.

On the other hand, in the examples, all percentages are by weight unless otherwise indicated, and the temperature is in degrees Celsius unless otherwise indicated, and the pressure is atmospheric pressure unless otherwise indicated. EXAMPLES

Analytical methods Determination of beta-qlucan content

The beta-glucan content is determined using an enzymatic method, according to a method adapted from the Megazyme kit method (reference K-EBHLG). The validation of the method carried out using the software E-noval (Arlenda) calculated an uncertainty of ± 3% on the value and a limit of acceptance of ± 10% .:

1 - A quantity of 20 mg +/- 0.1 mg of beta-glucan powder is suspended in a volume of 0.4 ml of 2N potassium hydroxide solution with stirring for 30 minutes in an ice-bath.

2- The pH of the suspension is then adjusted to 4.0 to 4.5 by adding a 1M sodium acetate solution at pH 7.

3- The mixture is incubated with a mixture of enzymes exo-1,3-glucanase, endo-1,3,3-glucanase, β-glucosidase and chitinase suspension (Megazyme) for 16 hours at 40 ° C.

4- After dilution and centrifugation, an aliquot is collected to determine the glucose content with the reagent GOPOD (glucose oxidase plus peroxidase and 4-aminoantipyrine compound diluted in buffer composed of p-hydroxybenzoic acid and sodium azide) .

An external calibration is established with a solution of glucans derived from yeasts, Megazyme) concentration 0, 5, 10, 15, 20 and 25 mg in a volume V of 2N sodium hydroxide solution and having undergone steps 1 to 4 ci -above.

The beta-glucan content is determined by measuring the absorbance by UV spectrometry, compared with the calibration curve.

The beta-glucan content is expressed in g of beta-glucan per 100g of wet beta-glucan. Determination of the water content

The loss on drying is determined using a thermodynamic method based on the European Pharmacopoeia 2.2.32 ("Loss on drying") method, with an accuracy of ± 0.15% of the value. The change from the Pharmacopoeia method concerns the choice of equipment (moisture analyzer instead of oven), without this having a significant impact on the value and the achievement of the results. Briefly, a known amount of beta-glucan powder is heated to 105 ° C, and mass loss is measured continuously using a calibrated moisture analyzer (Ohaus MB 45) until it reaches a value less than 1 mg per 90 seconds. When this value is reached, the weight of the loss on drying is calculated by subtracting the value of the dry matter from the total mass.

The water content is expressed in g of water per 100g of wet beta-glucan.

Determination of ash content

The method of analyzing the ash content is based on that of the

European Pharmacopoeia 2.4.16. A porcelain crucible is weighed. A known amount of beta-glucan is placed in the porcelain crucible and heated for 10h at 600 ° C in a calibrated muffle furnace (Carbolite, 201). After combustion, the porcelain crucible containing the beta-glucan of the sample is weighed. The ash content is expressed in g of water per 100 g of wet product. The ash content is expressed in grams of ash per 100 grams of wet product.

Determination of protein content

The protein content is determined according to a method based on total protein hydrolysis and spectrophotometric assay, according to the steps:

1 - 1 g of beta-glucan powder is suspended in a volume of 10 ml of concentrated sodium hydroxide solution (10N) prepared from sodium hydroxide R (European Pharmacopeia), at 130 ° C. for 90 minutes. minutes.

2- The solution of beta-glucan thus hydrolysed is then neutralized to pH 6.9-7.1 and the total volume adjusted to 50ml.

3- 100 μΙ of this solution are removed and 1 ml of 0.5 M acetate buffer at pH 5.1 are added.

4 ml of ninhydrin / hydrindantine solution (500 mg of ninhydrin and 150 mg of hydrindantine in 100 ml of ethylene glycol monoethyl ether) is then added to the solution prepared in step 3 (1.1 ml) to give form an absorbing complex at wavelength 570nm.

5. The absorbance of this solution is measured spectrophotometrically with a spectrophotometer calibrated according to the European Pharmacopoeia 2.2.25 ("absorption spectrophotometry, ultraviolet and visible"). An external calibration curve is established with a solution of bovine serum albumin (BSA, BioChemika, fraction V, batch No. S41084, Fluka) at concentrations 0, 2, 4, 6 and 8 mg which have undergone the same steps 1 to 5 as beta glucan.

The protein content is expressed in g of water per 100g of wet product.

Validation of the method using the E-novalet software gives an uncertainty of the method of ± 10% of the value and a limit of acceptance of ± 25%.

Determination of lipid content

The determination of the amount of lipids is based on Regulation (EC) No 152/2009 of 27-01-2009. The uncertainty on the value is ± 0.7%. The lipid content is expressed in g of water per 100 g of wet product.

Characterization of proportions in sugar-type repeat unit

The composition of the sugars is carried out by gas chromatography of the sugars after solubilization by total hydrolysis of beta-glucan and derivatization of the sugars according to the methods described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15 and York, et al. (1985) Methods Enzymol. 1 18: 3-40.

1 - 0.3 mg of beta-glucan powder are methanolysed with 1M hydrochloric acid in methanol at 80 ° C. for 16 hours.

2- The samples are then treated by treatment with Tri-Sil (Pierce) at 80 ° C for 30 minutes.

The resulting derivatives (per-O-trimethylsilyl-TMS) are analyzed by gas chromatography in comparison with an external calibration curve made with a standard for each sugar.

The sugar content is expressed in g of sugar per 100 g of wet beta-glucan which has been solubilized by hydrolysis.

Characterization of the sequences between the repetition units

The analysis of the glycosidic linkages between the glucose units was carried out according to the method described by York et al (1985) Methods Enzymol. 118: 3-40).

1 - 3mg of beta-glucan powder are solubilized in dimethylsulfoxide

2- The samples are then partially methylated according to the method described by Ciukan and Kerek (1984) in Carbohydr. Res. 131. -209-217, with butyllithium and methyl iodide: the samples are treated with sodium hydroxide for 15 minutes followed by treatment with methyl iodide for 45 minutes (this process is realized twice). The complex is then hydrolysed by reaction with 2M trifluoroacetic acid (2 hours at 121 ° C.).

The complex is then acetylated using a trifluoroacetic acid / acetic anhydride mixture.

The complex concentration is determined by gas chromatography with detection by mass spectrometry, compared with an external calibration curve made with a standard for each sugar.

The glycosidic linkages between the glucose units are expressed in g per 100 g of each identified individual wet sugar (in this case, glucose).

Example 1 Preparation of the Mycelium of Aspergillus niger

Aspergillus niger mycelium is a by-product of citric acid fermentation.

After the fermentation step, the mycelium is passed through a pressure band filter to be pressed and washed. The mycelium at the outlet of the pressure band filter has a solids content of 18% by weight. The mycelium is then dried in a rotary dryer ("rotary dryer" type) and then in a pneumatic dryer ("flash dryer" type).

Composition of the mycelium

The Aspergillus niger mycelium complies with the specifications in Table 1. Table 1

The type of A. niger mycelium may influence the purity of chitosan. The example shown below shows the difference in yield and purity between two mycelium, one unwashed after harvesting citric acid (black), the other pressed / washed after harvesting citric acid (beige color). Table 2

Comparing chitosan, it is noted that a higher yield is obtained from the pressed / washed mycelium. In terms of purity, the color of chitosan obtained from unwashed mycelium is also darker. The turbidity of a 1% (v / m) chitosan solution in 1% acetic acid is also higher than that of a chitosan solution obtained from the pressed / washed mycelium. The glucan content when the pressed / washed mycelium is used can be decreased below 10% by weight relative to the total mass of chitosan. Example 2: Industrial co-preparation of chitosan and glucans

Initial tests were conducted in the laboratory.

The conditions of the following deacetylation reaction of the chitin contained in the mycelium were tested:

Table 3

According to these conditions, the following characteristics of chitosan and glucan can be obtained:

Table 4

Reference A B C

Mass of 2.35g dry 1 .95g dry 3g dry

chitosan

recovered (g)

Yield (%) 7.8% 6.5% 10.0%

Glucan (% m / m) 17.7% 26.3% 12.0%

Degree 32.7% 38.9% /

acetylation

(Mol%)

Viscosity 4.7 4.35 /

Yield in ND ND 6.54g

glucan It is noted that the deacetylation conditions have an impact on the yields of chitosan and glucan.

The results show that it is preferable to perform further deacetylation reaction conditions in terms of temperature and time to increase the chitosan yield.

The conditions that give the best yields of chitosan are therefore applied on an industrial scale in the following examples are: 6 hours of deacetylation, followed by a rise in temperature to 1 10 ° C which lasts 2 hours, followed maintaining this temperature for 6 hours, followed by transfer of the reaction mixture for 1 hour.

The purity of the chitosan obtained is in adequacy with targeted applications. In addition, these conditions make it possible to preserve a large part of the insoluble glucan and thus to be able to recover them.

For an industrial process, it is possible to use a conical mixer (of the "Conical Mixer" type) which is, for example, for the invention a conical reactor of 4 m ³ equipped with a jacket for heating the reaction medium up to 120 ° C. vs. The agitation of the mixture is provided by a worm mounted on an orbital arm which travels around the periphery of the reactor at a rate of about 2 revolutions / min. Qlucan / Chitosan Separation Industrial Equipment

Once the deacetylation has been carried out, it is washed with water to remove the soda. After these washing steps, the suspension is brought to pH 4 by addition of concentrated acetic acid. Chitosan is soluble and glucan is insoluble.

To separate the two fractions, various industrial equipment was tested. Preferably, a nozzle centrifuge is used, and in particular a high performance vortex nozzle separator type centrifuge ("BTUX"). Those skilled in the art optimize the settings of this equipment to most effectively separate the chitosan glucan, including taking into account the yield of chitosan and glucan, and their respective purities.

The "BTUX" nozzle centrifuge settings are made to concentrate the glucan sufficiently that a minimum portion of chitosan is in the insoluble fraction including glucan (Table 5). Table 5

The proportions are measured after centrifugation by determining the volumes in a test tube.

The percentage of insoluble in the filtrates corresponds to the amount of glucans that still remains in the chitosan solution.

In this example, the best setting for the "BTUX" is when the pump runs at 20% of the maximum speed, as indicated by the high glucan concentration (<95%) that reveals a limited loss of chitosan. The last fraction of glucan present in the chitosan solution (15%) is separated by passage in a "sedative-type" equipment. The speed of the pump is adjusted according to the amount of insoluble present in the soluble fraction (chitosan) and the concentration of insoluble fraction (glucan). A proportion of insoluble in the filtrates is aimed at between 8 and 12%. This setting makes it possible to have a low content of soluble fraction in the low insoluble fraction (<5%).

For an industrial process, a nozzle centrifuge can also be used. The nozzle centrifuge is a solid / liquid separation equipment. The solid / liquid suspension is separated by the high speed rotation (HFA 8000 rpm / BTUX 7000 rpm) of a bowl that separates the fine solid particles from the liquid phase and concentrates them via nozzles. This high speed separation is accentuated thanks to a high filtration surface (plates).

In order to increase the glucan yield, the soluble fraction containing chitosan and optionally residual glucan is then passed through a high centrifugal separation separator, such as a "sedative-type" apparatus.

The Sedicer / Decanter is a solid / liquid separation equipment. The solid / liquid suspension is separated by the high speed rotation (sedimentant 4800 rpm / decanter 3500 rpm) of a bowl that separates the fine solid particles from the liquid phase and evacuate them with a worm placed inside this bowl. Adjustments are therefore made to optimize the recovery of glucan without significantly affecting the soluble fraction comprising chitosan from a purity and yield point of view. Example 3: Recovery of glucan

Objective: The conditions of this treatment make it possible to recover all the glucans at the outlet of the "BTUX" and the "Sedicant". High productivity with acceptable purity (glucan content> 60%) is targeted.

The choice of the precipitation mode has a very important impact on the productivity of the production line and on the purity of the glucan. Indeed, a precipitation with sodium hydroxide only does not allow easy separation in the filter press.

To promote separation, precipitation using lime milk has been tested. This type of additive makes it possible to obtain flocs (by flocculation). This type of precipitate texture allows for easier separation in industrial equipment and an advantageous reduction in separation time.

Preferably, the sodium hydroxide / lime mixture is used to effect flocculation of the glucan at a pH of about 10.

The proportion of soda and lime in the recovered mixture was varied on a laboratory scale, in order to verify how to limit calcium intake in final glucan (Table 6).

Table 6

The dryness corresponds to the percentage by mass of dry matter relative to the total mass of the cake recovered after concentration, typically with a filter press. At the laboratory scale, the conditions of the test N ° 1 are the best: 1 .6g of

NaOH and 4.2 g of CaO (mass ratio of approximately 1: 2.5).

The resulting product has a content of less than 10% ash, which is suitable for animal nutrition.

It is important to eliminate the aqueous phase sufficiently before passing through the pneumatic dryer ("flash dryer") to obtain a high glucan purity. .

The glucans are then concentrated in a filter press, or decanter, or band filter, or basket centrifuge in order to be able to proceed with their drying. Industrially, it is preferred to carry out the concentration using a filter press:

The filter press is a solid / liquid separation equipment. It allows to separate, under pressure, a suspension by frontal filtration of the solid particles through a filter medium (synthetic fabrics) held between two rigid trays.

The space 2 rigid trays makes it possible to harvest the dehydrated solid part thanks to the internal pressure of the filter. The liquid clarified portion is recovered via a cross tube collecting the filtrate of each filter medium. The concentrated solution is dried by a pneumatic dryer ("flash dryer").

The "flash dryer" is a drying equipment that can recover a fine powder from a moist and compact product. The wet product is fed via a worm into the drying chamber. In this drying chamber, a flow of hot air entrains and dries the solid particles and carries them through a classifier. The speed of rotation of the classifier makes it possible to control the size of the dry particles, if they are too large, they fall back into the drying chamber equipped with a grinder, are crushed and won by the flow of air. Once the classifier is passed, the airflow and the particles are separated in a cyclone. The fine powder is recovered under the cyclone and the air is filtered through a bag filter to be discharged to the outside.

Example 4: Industrial production of beta-glucan suitable for animal feed

In this example, 2.77m ³ of glucan was treated after separation in the nozzle centrifuge (BTUX type). This solution of glucan with a pH between 3.5 and 4.8 (acetic acid) is treated by addition of soda and lime so as to reach a pH greater than 10 (weight ratio of soda / lime of 1 / 5.6 (121 of sodium hydroxide at 30% and 901 lime at 30%, ie 4.8 and 27 kg respectively.) Soda is added to allow a pH greater than 4.8 to be obtained, then the lime is added in order to obtain a precipitate in the form of floc (pH 10).

The glucan thus precipitated was concentrated by a filter press in order to eliminate the water and to obtain the glucan in solid form at approximately 20% of dry matter. After this concentration step, the dryer ("flash dryer") is fed to recover 155 kg of a reference glucan powder L15.

The characteristics of the glucan thus produced are described in Table 7. Table 7

Example 5: Additional purification of glucan on an industrial scale

Glucan is resuspended in water after being precipitated and concentrated. The pH is adjusted between 6 and 7 by adding acetic acid to solubilize the chitosan still present. The suspension is sent for separation, for example in a filter press.

In this example, 4m ³ of glucans were treated after separation in the nozzle centrifuge (BTUX type). This solution of glucans with a pH between 3.5 and 4.8 (acetic acid) was treated by adding sodium hydroxide and lime so as to reach a pH greater than 10 (151% of 30% NaOH and 70% of lime). Glucan thus precipitated was injected into a filter press in order to be concentrated. The glucan was resuspended in a tank with 10m ³ of osmosis water. The pH of the suspension was adjusted to 6.4 with a volume of 171 of 80% acetic acid.

The suspension was reinjected into a filter press in order to be concentrated. The concentrated glucan was dried in a flash dryer. It was recovered 150kg of reference glucan L37.

The characteristics of the glucan thus produced are described in Table 8.

Table 8

L37

Glucan (w / w%) 90

Water (% w / m wet) 3.12

Ash (% w / m wet) 1 .5

Protein (w / w%) 0.21

Lipids (% m / m wet) 4.1 Example 6 Preparation and characteristics of batches of glucan used for the in vivo study of immunomodulatory properties

The glucans used for the studies are described in Table 9. The glucan of A. niger origin of reference L1 1 is that of example 4. The glucan resulting from yeast of reference "glucan" is a commercial product intended for the animal feed.

The A. niger glucan of reference L15 is prepared in the following manner, on an industrial scale:

3m ³ of insoluble fraction at the exit of BTUX (step iii) are harvested. The glucan solution was treated by addition of 50I of 30% sodium hydroxide so as to reach a pH greater than 1 1. The treated glucans were injected into the filter press in order to be concentrated (removal of water).

In order to lower the ash content (sodium hydroxide), 3m ³ of osmosis water was injected into the filter press. The glucans in solid form were then dried with a flash dryer, and 70 kg of reference glucan powder L15 are harvested.

Table 9

EXAMPLE 7 In Vivo Study of the Effect of Beta-Glucan Derived from A.niger on the Function of the Innate Immune System of Adult Pimephales Panchalae Promelas in the Absence of Stress and Under Stress

The Pimephales promelas fish is commonly used as a model for toxicological and immunological studies (Russom et al In: Environmental Toxicol Chem 16: 948 (1997)) Its innate cellular immune system is representative of the innate immune system of many animal 'man. Two beta-glucans from A.niger and produced as described in Examples 4 and 6 (references L1 1 and L15) are incorporated into a powdered fish feed, as described by Palic et al in: Developmental Comparative Immunol 30: 817 (2006), at doses of 5g / kg and 4g / kg respectively. Beta-glucan derived from yeast ("glucan") is incorporated in fish feed at a dose of 5 g / kg.

Adult fish Pimephales promelas are divided into several tanks according to the food they receive: control (food only) or food supplemented with one of the two beta-glucans, from A.niger (references L1 1 and L15) and a beta- glucan from yeast (reference "glucan"), or with myristate acetate phorbol (PMA, positive control).

The fish are fed every day. For half of the fish in each tank, stress is then applied 7 days after the start of the study, according to a method that mimics a fish handling and their excess, as described by Palic et al. in: Aquaculture 254: 675 (2006). Indeed, stress causes a loss of effectiveness of the innate immune defenses, in particular neutrophil functions such as for example the "oxidative burst" (massive emission of active form of oxygen) after stimulation with PMA and the degranulation of primary granules. The objective of this test is to verify that supplementation of fish with beta-glucans from A.niger stimulates neutrophil activity on the one hand, and that pre-supplementation with beta-glucans from A.niger avoids the reduction of immune defenses linked to stress on the other hand.

The marker for neutrophil activity is the effect on degranulation of primary neutrophil granules, which is determined by the release of myeloperoxidase (MPO) from the primary granules of neutrophils that were isolated from the kidneys of fish and stimulated by ionophore calcium, according to the method described by Palic et al. in: Developmental Comparative Immunol 30: 817 (2006). Fish supplementation with the 3 types of beta-glucan (L1 1-5%, L15-4% and L4-5%) is first studied for 21 days in normal conditions, without stress: it leads to increased degranulation compared to control (Figure 3). The effects of the two glucans from A.niger (L1 1 and L15) are comparable to those of glucan from yeast ("glucan"). Then, the effect of fish supplementation by the 3 types of beta-glucan on the stress response applied to the seventh day is studied: it leads to an increase in degranulation of neutrophils compared to the "stressed" control group after stress application (Figure 4). This result confirms that beta (1, 3) glucans from A.niger are bioactive and immunostimulating at doses administered to fish, at a level comparable to that of a beta-glucan derived from yeast.

EXAMPLE 8 Ex Vivo Study on Human Blood Cells

8.1 .- Beta-glucan used for this study

The Beta-glucan compound from A.niger used for this study is a grade for use in human nutrition.

This grade can be characterized as follows (Table 10):

Table 10

The sample was treated before carrying out the study, in particular to eliminate bacterial endotoxins potentially present: The sample is ground to obtain a particle size of about 70μηι (ά (0.9) <70μιτι). It was then sterilized by ethylene oxide and then treated with NaOH to eliminate bacterial endotoxins, endotoxins being known to stimulate the immune system.

8.2 - Activation of immune cells

Blood collected from healthy volunteers was exposed for 24 hours to increasing amounts of glucan (grade according to Example 8.1) ranging from 0.1 mg / mL to 1000 mg / mL. The activation of immune cells was then measured using cell membrane markers, in particular CD54, by flow cryometry. A clear activation of monocytes (FIG. 5) and plasmacytoid dendritic cells (FIG. 6) was observed by beta-glucan according to the present invention, and all the more so when beta-glucan is used at 100 μg / ml.

8.3 - Induction of cytokines

Blood collected from healthy volunteers was exposed for 24 hours to increasing amounts of glucan ranging from 0.1 mg / mL to 1000 mg / mL.

Cytokine production in the medium was then measured by multiplex. The glucan according to the present invention is capable of inducing the production of Interleukin 6 (IL-6) (FIG. 7) and Tumor Necrosis Factor alpha (TNF-α) (FIG. 8), in particular starting from 100 μg / ml. as well as Macrophage Inflammatory Protein 1 alpha (MIP-1 oc) from 1 g / ml (Figure 9). The production of other cytokines, such as Granulocyte Colony Stimulating Factor (G-CSF), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 1 beta (IL-1β), is also increased in the presence of beta-glucan. according to the present invention, and this by following a dose-response effect.

Glucan according to the present invention allows the activation of the immune cells of a human being and induces the production of cytokines, thereby demonstrating its benefit for use as an active agent in an immunostimulatory pharmaceutical composition of a human.

Example 9 - Cosmetic Formulations

The glucan used in the following examples has a composition according to Example 8, without subsequent treatment for the elimination of bacterial endotoxins potentially present.

In order to prepare formulations that are acceptable in cosmetics, ie for topical application, the particle size distribution of beta-glucan is preferably as follows:

The diameter of 90% (d (0.9)) of the particles is less than 350 microns, preferably less than 100 microns for oil-in-water and water-in-oil emulsions, and preferably less than 50 microns for reds. lip balms and lip balms (measured by laser diffractometry). EXAMPLE 9.1 - Anti-aging moisturizing composition containing beta-glucan from A niger.

This formulation has the following composition:

Table 11

qs how su cient to reach

Procedure

1. Mix the components of phase A, and melt approximately at eO '

2. Dissolve the ingredients of phase B together

3. Add components from phase C to mixture 1 while stirring

4. Add mixes 2 and 3 while stirring at 250 rpm, let cool

5. Add phase D to mixture 4 at 35 ° C

6. Adjust the pH of Mix 5 with Phase E to a value between 6.0 and 6.5

7. Finish by homogenizing.

EXAMPLE 9.2- Baby lotion containing beta-glucan from A niger.

This formulation has the following composition Table 12

Procedure

1. Mix the components of phase A, and melt at approximately 80 ° C

2. Mix the ingredients of phase B

3. Add phase C to mixture 2 and mix with vigorous stirring until a clear and homogeneous solution is obtained 4. Add phase D to mix 3 while mixing well and heating to 80 ° C

5. Add phase E to mix 4 and mix until a homogeneous solution is obtained

6. Add phase F to mix 1 and mix

7. Add mixture 5 to mix 6 and mix at 300 rpm until temperature decreases

8. Adjust the pH with phase G to 5.5

9. Finish by homogenizing.

Results

pH: 5.45

Viscosity (Brookfield, 20 ^< C at 20 rpm): 4000 mPa.s

Stability: satisfactory after 12 weeks at room temperature (20 ° C), 40 ° C and 45 ° C

EXAMPLE 9.3- Moisturizing cream containing beta-glucan from A niger. This formulation has the following composition:

Table 13

E Citric acid 10% q.s.

Procedure

1. Mix the components of phase A, and melt approximately at δΟ 'Ό

2. Dissolve the ingredients of phase B

3. Add components from phase C to mixture 1 while stirring

4. Add mixes 2 and 3 while stirring at 250 rpm, let cool

5. Add phase D to mixture 4 at 35 ° C

6. Adjust the pH of Mix 5 with Phase E to a value between 6.0 and 6.5

7. Finish by homogenizing.

Results

pH 6.30

Appearance: Creamy white cream

Viscosity (Brookfield, 20 ^< 20 rpm): 40300 mPa.s

Stability: satisfactory after 12 weeks at room temperature (20 ° C), 40% and

45 ° C

Claims

1. - Method for the preparation of glucan from Aspergillus niger characterized in that it comprises (i) at least partial deacetylation of Aspergillus niger mycelium; (ii) acid treatment of the deacetylated or partially deacetylated mycelium, preferably after purification and / or washing, to obtain insoluble glucan and soluble chitosan, said acidic treatment comprising contacting the deacetylated mycelium with an acidic solution; (iii) the separation of soluble chitosan on the one hand, and insoluble glucan on the other hand; (iv) an alkaline treatment of glucan comprising contacting glucan with an alkaline solution to flocculate glucan; and (v) drying the flocculated glucan to obtain a glucan powder.

2. - Method according to claim 1, characterized in that the alkaline solution of step (iv) comprises or consists of a mixture of sodium hydroxide (NaOH) and calcium hydroxide (Ca (OH ) ₂ ).

3. - Method according to any one of the preceding claims, characterized in that step (iv) comprises a weight ratio sodium hydroxide / calcium hydroxide of 1/2 to 1/10, between sodium hydroxide and calcium hydroxide.

4. - Method according to any one of the preceding claims, characterized in that the separation is carried out by a continuous centrifugal separation nozzle.

5. - Method according to any one of the preceding claims, characterized in that it comprises an additional step (iiia) of treatment of the soluble chitosan obtained in step (iii) to separate the insoluble glucan, which is added insoluble glucan recovered in step (iii).

6. - Method according to any one of the preceding claims, characterized in that the deacetylation is carried out by bringing the mycelium into contact with an alkaline material, and preferably sodium hydroxide (NaOH), at a concentration of a temperature, and for a time sufficient to deacetylate the chitin to chitosan with a minimum yield of 4% in chitosan and a degree of deacetylation of 0 to 50%, and preferably 10 to 25%.

7.- Method according to any one of the preceding claims, characterized in that prior to step (ii) the pH is decreased by one or more washings with water.

8.- Method according to any one of the preceding claims, characterized in that the acidic treatment of step (ii) comprises the addition of an organic acid until a pH of between 3 is obtained. and 5.5, and preferably between 3.5 and 4.5.

9. - Method according to any one of the preceding claims, characterized in that the acid treatment of step (ii) comprises or consists of the addition of acetic acid to the deacetylated mycelium obtained in step (i) .

10. - Method for co-preparation of chitosan and glucan from Aspergillus niger, characterized in that it comprises the glucan preparation according to any one of the preceding claims, and the chitosan preparation.

1 1. - Beta-glucan from Aspergillus niger obtainable by a method as defined in any one of the preceding claims.

12. Beta-glucan from Aspergillus niger, according to claim 1 1, characterized in that it is suitable for animal feed.

13. - Beta-glucan from Aspergillus niger, according to claim 1 1, characterized in that it is suitable for application in humans.

14. Beta-glucan from Aspergillus niger for the modulation of the immune system of a human being or an animal.

15. Beta-glucan from Aspergillus niger according to claim 14, characterized in that it is intended for improving the functions of the innate immune system, in particular the functions of neutrophils.

16.- dietary supplement composition for humans or animals, characterized in that it comprises beta-glucan from Aspergillus niger as defined in any one of claims 1 1 to 15.

17.- Solid food for fish, characterized in that it comprises betaglucan from Aspergillus niger as defined in any one of claims 1 1 to 15.

18. A pharmaceutical composition comprising beta-glucan derived from Aspergillus niger as defined in any one of claims 1 1 to 15.

19. - An immunostimulatory pharmaceutical composition comprising beta-glucan from Aspergillus niger as defined in any one of claims 1 1 to 15.

20. - Cosmetic composition comprising beta-glucan from Aspergillus niger as defined in any one of claims 1 1 or 13.