ES2390063B1 - Glycosaminoglycans with very low methanol content. - Google Patents

Glycosaminoglycans with very low methanol content. Download PDF

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Publication number
ES2390063B1
ES2390063B1 ES201130576A ES201130576A ES2390063B1 ES 2390063 B1 ES2390063 B1 ES 2390063B1 ES 201130576 A ES201130576 A ES 201130576A ES 201130576 A ES201130576 A ES 201130576A ES 2390063 B1 ES2390063 B1 ES 2390063B1
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glycosaminoglycan
water
ppm
microwave
hyaluronic acid
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ES2390063A1 (en
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Ramón Ruhí Roura
Carmen HERNÁNDEZ PÉREZ
Josep Escaich Ferrer
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Bioiberica SA
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Bioiberica SA
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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G
    • C08J3/00Processes of treating or compounding macromolecular substances
    • C08J3/28Treatment by wave energy or particle radiation
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G
    • C08J2305/00Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G
    • C08J2305/00Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Abstract

Glycosaminoglycans with very low methanol content. # The present invention relates to a process for preparing a glycosaminoglycan with a methanol content of less than or equal to 10 ppm, in which water is sprayed on the glycosaminoglycan with stirring and irradiated with microwave under vacuum at a temperature below 70 ° C. The process allows to formulate said glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, in a product suitable for use in a food supplement or in a functional food.

Description

Glycosaminoglycans with very low methanol content.

Technical sector of the invention

The present invention relates to a process for preparing a glycosaminoglycan with a methanol content of less than or equal to 10 ppm. The process allows to formulate said glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, in a product suitable for use in a food supplement or in a functional food.

Background of the invention

Glycosaminoglycans (GAG) are polymeric biomolecules of high molecular weight that consist of a repeated dimeric structure. They are found primarily in living organisms, where they develop different physiological functions. Glycosaminoglycans include chondroitin sulfate, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate and queratane sulfate. Except for hyaluronic acid, all others contain sulfate groups in their structures.

Chondroitin sulfate has a polymeric structure characterized by a repeating disaccharide, consisting of N-acetyl-D-galactosamine and D-glucuronic acid. Most N-acetyl-D-galactosamine residues are sulfated. Generally, it has a molecular weight between 10,000 and 70,000 Da, depending on the source and procedure for obtaining it (N. Volpi, Journal of Pharmaceutical Sciences, 96 (12), 3168-3180, (2007)). The utility of chondroitin sulfate in the treatment of cardiovascular diseases (US 3,895,106), psoriasis (WO2005 / 014012) or osteoarthritis (MG Lequesne, Rev. Rhum. Eng. Ed., 61, 69-73 has been described (1994); G. Verbruggen et al., Osteoarthritis Cart., 6 (Supplement A), 37-38 (1998)).

Hyaluronic acid is a non-sulfated glycosaminoglycan, with a polymeric structure characterized by a repeating disaccharide, consisting of N-acetyl-D-glucosamine monosaccharides and D-glucuronic acid. It is one of the main components of cartilage, synovial membrane and synovial fluid. Particularly important is its use in the treatment of osteoarthritis, usually intra-articularly. Its use has also been described in ophthalmology, to accelerate wound healing, as well as in cosmetics.

Chondroitin sulfate glycosaminoglycans, hyaluronic acid, dermatan sulfate and queratane sulfate are used both in nutrition, in the form of food supplements or functional foods, as in pharmaceutical preparations.

In large part of the preparation procedures for chondroitin sulfate glycosaminoglycans, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate and keratane sulfate, from animal tissues (trachea, mucosa, crest, etc.), It uses methanol as a solvent, since the use of undenatured ethanol has a high cost. The amount of residual methanol of the glycosaminoglycans obtained through some of the procedures described in the literature is approximately 50-2,000 ppm.

The health authorities allow up to 3,000 ppm of methanol in an active substance intended for the preparation of a medicine (ICH Harmonized Tripartite Guideline. Impurities: Guideline for Residual Solvents Q3C (R5)). However, due to the toxicity of methanol, since it is difficult to control the amount of food supplement or functional food that a living being could ingest, the authorities are stricter when the compound, in this case a glycosaminoglycan, is used in nutrition , allowing only a maximum amount of methanol of 10 ppm (Directive 2009/32 / EC of the European Parliament and of the Council, April 23, 2009).

Microwaves are a form of electromagnetic energy with frequencies between 300 MHz and 300 GHz, generated by magnetrons under the combined force of an electric field and a magnetic field, perpendicular to each other. The most used frequencies are 915 MHz and 2,450 MHz.

One of the best known applications of microwaves is the microwave oven, which uses a magnetron to produce waves at a frequency of approximately 2,450 MHz. These waves make the water molecules vibrate or rotate, generating heat. Because most foods contain a significant percentage of water, they can be easily heated or cooked in this way.

Microwaves are used in various industrial sectors, such as in the food, ceramics, rubber, paper, and pharmaceutical industries.

Microwave drying under vacuum is the result of the combination of two technologies: microwaves that provide fast and uniform drying, allowing heat to be generated evenly inside the solid and vacuum that allows drying temperatures to be reduced. It is widely used in the food industry, for example, to dehydrate fruits and vegetables (see, for example, patent application WO2009 / 066259; patent application US2008 / 0179318) and in the pharmaceutical industry, for example, to dry and / or granular active ingredients (M.

N. Berteli et al., Braz. J. Chem. Eng., 26 (2), 317-329 (2009); G. Farrel et al., Drying Technology, 23 (9-11), 2131

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2146 (2005); C.M. McLoughlin et al., Drying Technology, 21 (9), 1719-1733 (2003)) or to dehydrate temperature sensitive biological materials (patent application WO2010 / 124375).

In view of the above, it is of great interest to find a method of preparing a glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, with a methanol content of 10 ppm or less, so that it can be used to prepare a functional food or a food supplement, complying with the requirements of the competent authorities.

Until now, a process for preparing a glycosaminoglycan, for example chondroitin sulfate or hyaluronic acid, with an amount of methanol less than or equal to 10 ppm, starting from a glycosaminoglycan obtained by means of a process in which used methanol as a solvent

Explanation of the invention.

The present inventors have found that, surprisingly, the process of the present invention allows to prepare a glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, with a methanol content of less than or equal to 10 ppm. When in the present invention water is sprayed, under stirring, onto a glycosaminoglycan containing an amount of methanol greater than 10 ppm and subjected to the microwave action under vacuum, surprisingly, a glycosaminoglycan with an amount of methanol less than or equal is obtained at 10 ppm. Therefore, the glycosaminoglycan thus obtained can be used in the preparation of food supplements or functional foods.

Thus, the present invention relates to a process for preparing a glycosaminoglycan with a methanol content of less than or equal to 10 ppm, comprising the following steps:

a) dispose of a glycosaminoglycan with a methanol content greater than 10 ppm in a container of an equipment that incorporates a microwave and a vacuum pump;

b) spray water on the glycosaminoglycan of step a), under stirring, and

c) subjecting the glycosaminoglycan of step b) to microwave radiation, under vacuum and stirring, at a temperature below 70 ° C and for a period of time that allows obtaining the glycosaminoglycan with a methanol content of 10 ppm or less. To control the temperature during this stage, water can be circulated through the outer jacket of the container containing the glycosaminoglycan.

In a preferred embodiment, the process comprises an additional stage before step b), in which the glycosaminoglycan of step a) is subjected to microwave radiation, under vacuum and stirring, at a temperature below 70 ° C and during a period of time to obtain a glycosaminoglycan with a methanol content between 10 ppm and 150 ppm, preferably 100 ppm or 88 ppm. To control the temperature during this additional stage, water can be circulated through the outer jacket of the container containing the glycosaminoglycan.

In another equally preferred embodiment, in step c), when the temperature reaches 70 ° C, (i) the microwave is stopped; (ii) the vacuum is broken; (iii) the glycosaminoglycan is cooled; (iv) water is sprayed, and (v) the microwave and vacuum are reconnected. This cycle of stages (i), (ii), (iii), (iv) and (v) can be repeated several times until a methanol content of 10 ppm or less is reached.

In another equally preferred embodiment, in step c), (i) the microwave is stopped; (ii) the vacuum is broken;

(iii) water is sprayed, and (iv) the microwave and vacuum are reconnected. This cycle of stages (i), (ii), (iii) and (iv) can be repeated several times until a methanol content of 10 ppm or less is reached.

In another equally preferred embodiment, the glycosaminoglycan is in powder form.

In another equally preferred embodiment, the methanol content of the glycosaminoglycan of step a) is greater than 150 ppm, preferably between 151 ppm and 2,000 ppm, more preferably 198 ppm or 300 ppm.

In another equally preferred embodiment, the microwave operates at a power comprised between 10 and 70 watts per kg of glycosaminioglycan of stage a), preferably at 40 or 42 watts per kg of glycosaminioglycan of stage a).

In another equally preferred embodiment, the vacuum or vacuum pressure is 50 mbar or 70 mbar.

In a particularly preferred embodiment, when water is sprayed, the amount of water that is sprayed is between 1% and 3% by weight of water relative to the weight of the glycosaminoglycan of step a), preferably, the amount of water that is sprayed it is 1.5% by weight of water with respect to the weight of the glycosaminoglycan of step a).

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In another particularly preferred embodiment, the glycosaminoglycan of step c) is formulated in a product suitable for use in a food supplement or in a functional food.

In another particularly preferred embodiment, the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, queratane sulfate and mixtures thereof. Preferably the glycosaminoglycan is chondroitin sulfate or hyaluronic acid.

The present invention also relates to a glycosaminoglycan, preferably chondroitin sulfate, with a methanol content of less than or equal to 10 ppm, obtainable by the process described above.

The present invention also relates to a glycosaminoglycan, preferably chondroitin sulfate, with a methanol content of less than or equal to 10 ppm, obtainable by the procedure described above, suitable for use as a food supplement or functional food.

The glycosaminoglycans that are used in the process of the present invention as starting compounds can be obtained from various animal tissues, such as, for example, crow's crests, swine trachea, bovine trachea, porcine mucosa, bovine mucosa, skin, sternum of a bird or elasmobranch fish skeleton, such as the shark, through procedures described in the literature, using methanol or ethanol denatured with methanol as solvent. For example, chondroitin sulfate can be obtained by proteolytic enzymatic digestion of cartilaginous tissues of animals, for example tracheas of cattle or pigs. Hyaluronic acid can be obtained from rooster crests, which once chopped are digested with a proteolytic enzyme. The enzyme is subsequently deactivated by heating, filtered, the dermatan sulfate is removed and the hyaluronic acid is precipitated with a methanol-acetone mixture. It is anhydrified with methanol and then dried and ground.

All starting glycosaminoglycans used in the present invention contain an amount of methanol greater than 10 ppm.

Chondroitin sulfate from cartilaginous tissue is found mainly in two isomeric forms, which differ in the position of the sulfate group present in the N-acetylgalactosamine residue, chondroitin 4-sulfate (chondroitin sulfate A; R1 = SO3- Na +, R2 = H) and chondroitin sulfate (chondroitin sulfate C; R1 = H, R2 = SO3-Na +), which are represented by the following structure:

OR1

OR2

NaO 2 C

OR OR

HO

OR

OR

OR

OH NHAc

n

R1 = SO3-Na +, R2 = H

R2 = SO3-Na +, R1 = H

In addition, chondroitin sulfate may contain sulfate groups at positions 4 and 6 of the N-acetylgalactosamine residue, (chondroitin sulfate E), or at positions 6 of N-acetylgalactosamine and 2 of Dglucuronic acid (chondroitin sulfate D).

Hyaluronic acid is a non-sulfated glycosaminoglycan, of molecular weight between 100,000 daltons and 3,000,000 daltons. Its polymeric structure is characterized by a repeating disaccharide, consisting of N-acetyl-D-glucosamine and D-glucuronic acid:

OH

NaO 2 C

HO

OR OR

HO

OR

OR

OR

OH NHAc

n

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Due to the negative charges present in the molecule, both chondroitin sulfate and hyaluronic acid are in salt form, for example, in commercial preparations, chondroitin sulfate and hyaluronic acid are in the form of sodium salt.

When "ppm" is referred to in the present invention, reference is made to "parts per million." It is a unit of measurement of concentration, and refers to the amount of units of substance, in the present invention, methanol, which are per million units of the set, in the present invention of starting glycosaminoglycan.

The term "spray" of the present invention has the same meaning as the term "spray."

When "rpm" is referred to in the present invention, reference is made to "revolutions per minute".

When the present invention refers to "under agitation", reference is made to agitation that allows the glycosaminoglycan to be homogenized, especially when water is sprayed.

When "impeller" is referred to in the present invention, reference is made to a rotating impeller blade, stirring the glycosaminoglycan.

When "chopper" is referred to in the present invention, reference is made to a blade system that rotates at high speed, agitating the glycosaminoglycan and preventing the formation of large agglomerates.

To carry out the process of the present invention, equipment containing a microwave generator, a chamber with a container to house the glycosaminoglycan and capable of receiving microwave radiation produced by the generator, a vacuum system can be used capable of applying a vacuum to the chamber, an outer jacket to the container containing the glycosaminoglycan, through which water or other means can be circulated to keep the glycosaminoglycan at the desired temperature, stirring means, for example an impeller and a chopper that they can be introduced into the glycosaminoglycan to carry out good agitation and means to spray water. For example, a commercial mixer-granulator can be used, such as the Collette Ultima-Pro 25 L mixer-granulator or the Lbb Bohle VMA 300.

The microwave power can be, for example, 915 MHz or 2,450 MHz, although it is preferred to be 2,450 MHz.

In the stage in which it is irradiated, without spraying water, the speed of the impeller can be, for example, 50 rpm and that of the chopper of, for example, 600 rpm.

When water is sprayed, the speed of the impeller can be, for example, 200 or 220 rpm and that of the chopper, for example, 1,500 or 2,500 rpm.

The equipment vacuum pump must allow a vacuum or vacuum pressure of, for example, 50 mbar or 70 mbar. For example, a Watson MarloW pump can be used.

The water that is sprayed on the glycosaminoglycan can be water free of ions and microorganisms, and can be sprayed with a peristaltic pump.

The ppm of methanol of the starting glycosaminoglycan and glycosaminoglycan at the end of the process can be determined by gas chromatography.

From the procedures described in the literature, using methanol or ethanol denatured with methanol as solvent, it is not possible to obtain a glycosaminoglycan, for example chondroitin sulfate or hyaluronic acid, with a methanol content of 10 ppm or less. Generally, in the processes in which methanol or ethanol denatured with methanol is used as a solvent, the chondroitin sulfate obtained contains between 50 ppm and 2,000 ppm of methanol. The inventors of the present invention have found that if the methanol is removed from said chondroitin sulfate under vacuum, without spraying water, it is not possible to reduce the methanol content below 45 ppm; On the contrary, if water is stirred under stirring prior to vacuum microwave action, the methanol content is reduced to less than 10 ppm.

The inventors of the present invention have observed that in the process of the present invention, when water is sprayed on the glycosaminoglycan without good stirring, part of the product is dissolved forming lumps.

The inventors of the present invention have also found that it is important that during the process the temperature does not reach or exceed 70 ° C.

The present inventors have found that the reduction of the methanol content of a glycosaminoglycan below 10 ppm or up to 10 ppm takes place under certain conditions: microwave under vacuum, water spray, stirring and temperature below 70 ° C.

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Thus, the process of the present invention allows: (i) to prepare a glycosaminoglycan for food with a methanol content of less than or equal to 10 ppm, complying with the requirements established in the Directive of the European Parliament and (ii) using methanol or denatured ethanol with methanol in the process of obtaining a glycosaminoglycan for food, preferably chondroitin sulfate or hyaluronic acid, from animal tissues, which leads to a reduction in production costs.

To prepare both a food supplement and a functional food, glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, is formulated with suitable components and / or excipients used in nutrition. The food supplement may be in the form of tablets, capsules, solutions, suspensions or sachets. The functional food may be in the form of yogurts, milk, fermented milk, fruit juices, vegetable juices, soups, dehydrated foods, cookies or baby foods.

Brief Description of the Figures

Figure 1 represents the ppm of methanol over time in minutes, when a chondroitin sulfate is subjected to microwave radiation under vacuum without spraying water or spraying water. It is displayed after 375 minutes.

Figure 2 represents the ppm of methanol over time in minutes, when starting from a chondroitin sulfate with a methanol content of 43.8 ppm and subjected to microwave radiation under vacuum without spraying water or spraying water .

Detailed description of the preferred embodiments

Examples

The following examples are merely illustrative and do not represent a limitation of the scope of the present invention.

Example 1: Preparation of chondroitin sulfate with a methanol content of 8.3 ppm, from a chondroitin sulfate with a methanol content of 88 ppm, by microwave action under vacuum and water spraying

10 kg of chondroitin sulfate containing 88 ppm of methanol were introduced into the container of a microwave equipment with a frequency of 2,450 MHz (Mixer-granulator Collette Ultima-Pro 25 L) that had a vacuum pump incorporated. The chondroitin sulfate was stirred by means of the impeller (at 200 rpm) and the chopper (at 2,500 rpm) of the equipment, keeping the chondroitin sulfate at a temperature below 70 ° C, by means of a jacket outside the vessel through which water circulated . Then, and always under stirring, 150 g of water were sprayed (1.5% by weight of water with respect to the weight of the starting chondroitin sulfate), the vacuum was made until reaching 50 mbar and it was irradiated with microwave at a power of 40 watts per kg of the starting chondroitin sulfate (in total 400 watts). When the temperature reached 70 ° C, the microwave was stopped, the vacuum was broken, chondroitin sulfate was cooled by the water flowing through the outer jacket of the container containing it, 150 g of water was sprayed again, the vacuum up to 50 mbar and irradiated with microwave at a power of 40 watts per kg of the starting chondroitin sulfate (in total 400 watts). This cycle with the stages of stopping the microwave, breaking the vacuum, cooling the chondroitin sulfate, spraying water, vacuuming and irradiating with microwaves, was repeated every time the temperature reached 70 ° C. At the end of the cycle or cycles, the microwave was stopped, the vacuum was broken and the chondroitin sulfate with a methanol content of 8.3 ppm was recovered.

The ppm of methanol was determined by gas chromatography.

Example 2: Preparation of chondroitin sulfate with a methanol content of 6.4 ppm, from a chondroitin sulfate with a methanol content of 198 ppm, by microwave action under vacuum and water spraying

112.5 kg of chondroitin sulfate with a methanol content of 198 ppm were weighed and placed inside the vessel of a microwave equipment of 2,450 MHz frequency (Lbb Bohle VMA 300 mixer-granulator) that had a vacuum pump incorporated . Chondroitin sulfate was stirred by means of the impeller (at 50 rpm) and the chopper (at 600 rpm) of the equipment, keeping the chondroitin sulfate at a temperature below 70 ° C by means of a jacket outside the vessel, through which water circulated at 20 ° C. Then, the vacuum was made until it reached 70 mbar and was irradiated with microwave for 7 hours, at a power of 42 watts per kg of the starting chondroitin sulfate. After this time, the microwave was stopped, the vacuum was broken and the speed of the impeller (at 220 rpm) and the chopper (at 1,500 rpm) was increased, keeping the chondroitin sulfate at a temperature below 70 ° C, by means of outer jacket to the vessel through which water circulated. Then, and always under stirring, 1.68 kg of water (1.5% water weight with respect to the weight of the starting chondroitin sulfate) was sprayed, the vacuum was made until reaching 70 mbar and irradiated with microwave for 2 hours, at a power of 42 watts per kg of the starting chondroitin sulfate. After this time, the microwave stopped and the vacuum broke. Then, and always under stirring, 1.68 kg of water was sprayed, the vacuum was made until it reached 70 mbar and the microwave was connected, radiating for 2 hours, at a power of 42 watts per kg of

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starting chondroitin sulfate. In total, six cycles were carried out with the following consecutive stages per cycle: stop the microwave, break the vacuum, spray water, vacuum and radiate for 2 hours. After six cycles the microwave was stopped, the vacuum was broken and the chondroitin sulfate with a methanol content of 6.4 ppm was recovered.

This Example demonstrates that the procedure can be applied to prepare large amounts of chondroitin sulfate with a methanol content of less than 10 ppm.

Example 3: Study of microwave action under vacuum and without spraying water on a chondroitin sulfate with a methanol content of 198 ppm

The present Example was carried out to study the effect of microwave irradiation under vacuum without spraying water, in order to compare it with the process of the present invention, in which water is sprayed.

10 kg of chondroitin sulfate with a methanol content of 198 ppm were weighed and placed inside the container of a 2,450 MHz frequency microwave equipment (Collette Ultima-Pro 25 L mixer-granulator) that had a vacuum pump incorporated . Chondroitin sulfate was stirred by means of the impeller (at 50 rpm) and the chopper (at 600 rpm) of the equipment, keeping the chondroitin sulfate at a temperature below 70 ° C, by means of a jacket outside the vessel through which water circulated at 20 ° C. Then, the vacuum was made to reach 50 mbar and irradiated with microwave for 8 hours, at a power of 40 watts per kg of starting chondroitin sulfate (in total 400 watts). The temperature of the chondroitin sulfate contained in the equipment container was controlled throughout the procedure so that it did not exceed 70 ° C. After this time, the microwave was stopped, the vacuum was broken and the chondroitin sulfate with a methanol content of 88 ppm was recovered.

Example 4: Study of the removal of methanol from a sample of chondroitin sulfate, by microwave under vacuum. Effect of water spraying on methanol removal

The procedure of Example 3 was followed, but starting from 7 kg of chondroitin sulfate containing 588 ppm of methanol, and stopping the procedure for collecting samples several times. A chondroitin sulfate with a methanol content of 91.7 ppm was obtained. Figure 1 shows the procedure after 375 minutes. As can be seen, at 442 minutes a methanol ppm value of 98.7 was reached. By irradiating for an additional 26 minutes, it was only possible to lower to a methanol ppm value of 91.7.

Then, and starting from the above chondroitin sulfate containing 91.7 ppm of methanol, the procedure of Example 1 was applied, that is, water was sprayed and irradiated with microwave under vacuum. As can be seen in Figure 1, at 524 minutes (56 minutes with the conditions of Example 1) the methanol content was reduced to 33.3 ppm, and at 1,019 minutes (551 minutes with the conditions of Example 1) a chondroitin sulfate with a methanol content of 8.3 ppm was recovered.

The study was repeated, but this time starting from 7 kg of chondroitin sulfate that contained less methanol, specifically contained 43.8 ppm of methanol. By applying the procedure of Example 3 (without spraying water), the amount of methanol was reduced very little, since at 269 minutes it was only lowered to 41.3 ppm (see Figure 2).

Then, starting from the above chondroitin sulfate containing 41.3 ppm of methanol, the procedure of Example 1 was applied, that is, water was sprayed and irradiated with microwaves under vacuum. As can be seen in Figure 2, at 358 minutes (89 minutes under the conditions of Example 1) the methanol content was reduced to 26.9 ppm, and at 606 minutes (337 minutes under the conditions of Example 1) The methanol content had already been lowered to 10.8 ppm.

Taking these results into account, it can be concluded that to remove methanol from a glycosaminoglycan, this is a case of a chondroitin sulfate, up to an amount less than or equal to 10 ppm, water spraying is essential during the microwave irradiation procedure under vacuum

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Claims (17)

  1.  CLAIMS
    1. Process for preparing a glycosaminoglycan with a methanol content of 10 ppm or less, which comprises the following steps:
    a) dispose of a glycosaminoglycan with a methanol content greater than 10 ppm in a container of an equipment that incorporates a microwave and a vacuum pump;
    b) spray water on the glycosaminoglycan of step a), under stirring, and
    c) subjecting the glycosaminoglycan of step b) to microwave radiation, under vacuum and stirring, at a temperature below 70 ° C and for a period of time that allows obtaining the glycosaminoglycan with a methanol content of 10 ppm or less.
  2. 2. The method according to claim 1, comprising an additional stage before step b), wherein the glycosaminoglycan of step a) is subjected to microwave radiation, under vacuum and stirring, at a temperature below 70 ° C and for a period of time to obtain a glycosaminoglycan with a methanol content between 10 ppm and 150 ppm.
  3. 3. The method according to claim 1 or 2, wherein in step c), when the temperature reaches 70 ° C, (i) the microwave is stopped; (ii) the vacuum is broken; (iii) the glycosaminoglycan is cooled; (iv) water is sprayed, and
    (v) the microwave and vacuum are reconnected.
  4. 4. The method according to claim 1 or 2, wherein in step c), (i) the microwave is stopped; (ii) the vacuum is broken; (iii) water is sprayed, and (iv) the microwave and vacuum are reconnected.
  5. 5. The process according to any one of claims 1 to 4, wherein the glycosaminoglycan is in powder form.
  6. 6. The process according to any one of claims 1 to 5, wherein the methanol content of the glycosaminoglycan of step a) is greater than 150 ppm.
  7. 7. The process according to claim 6, wherein the methanol content of the glycosaminoglycan of step a) is comprised between 151 ppm and 2,000 ppm.
  8. 8. The method according to any one of claims 1 to 7, wherein the microwave operates at a power between 10 and 70 watts per kg of glycosaminioglycan of step a), preferably works at 40 watts per kg of glycosaminioglycan of stage a).
  9. 9. The method according to any one of claims 1 to 8, wherein the vacuum is 50 mbar.
  10. 10. The method according to any one of claims 1 to 9, wherein when water is sprayed, the amount of water sprayed is between 1% and 3% by weight of water with respect to the weight of the glycosaminoglycan of the stage to).
  11. 11. The method according to claim 10, wherein the amount of water that is sprayed is 1.5% by weight of water with respect to the weight of the glycosaminoglycan of step a).
  12. 12. The method according to any one of claims 1 to 11, wherein the glycosaminoglycan of step c) is formulated in a product suitable for use in a food supplement or in a functional food.
  13. 13. The process according to any one of claims 1 to 12, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, queratane sulfate and mixtures thereof.
  14. 14. The method according to claim 13, wherein the glycosaminoglycan is chondroitin sulfate.
  15. 15. The method according to claim 13, wherein the glycosaminoglycan is hyaluronic acid.
    ES 2 390 063 Al
    Without spraying water Spraying water
    Figure 1
    Without spraying water
    Spraying water
    Figure 2
    SPANISH OFFICE OF THE PATENTS AND BRAND
    Application no .: 201130576
    SPAIN
    Date of submission of the application: 04.13.2011
    Priority Date:
    REPORT ON THE STATE OF THE TECHNIQUE
    51 Int. Cl.: See Additional Sheet
    RELEVANT DOCUMENTS
    Category
    56 Documents cited Claims Affected
    TO
    WO 2006101030 A1 (Q P CORP) 28.09.2006, the whole document. 1-15
    TO
    JP 2008179710 A (SHISEIDO CO LTD) 07.08.2008, (summary) WPI [online databases] Derwent Publications LTD. [retrieved on 10.10.2012]. Recovered from Epoque. Accession No. 2008-L39641 [67]. 1-15
    TO
    WO 2007099830 A1 (Q P CORP) 07.09.2007, the whole document. 1-15
    TO
    EN 8800280 A1 (BIOIBERICA) 01.01.1988, the whole document. 1-15
    Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application
    This report has been prepared • for all claims • for claims no:
    Date of completion of the report 17.10.2012
    Examiner M. Hernández Cuellar Page 1/4
    REPORT OF THE STATE OF THE TECHNIQUE
    Application number: 201130576
    CLASSIFICATION OBJECT OF THE APPLICATION C07H5 / 00 (2006.01)
    C08B37 / 00 (2006.01) F26B5 / 04 (2006.01) Minimum documentation sought (classification system followed by classification symbols)
    C07H, C08B, F26B
    Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, MEDLINE, BIOSIS, EMBASE, CAPLUS
    State of the Art Report Page 2/4
     WRITTEN OPINION
    Application number: 201130576
    Date of Written Opinion: 17.10.2012
    Statement
    Novelty (Art. 6.1 LP 11/1986)
    Claims Claims 1-15 IF NOT
    Inventive activity (Art. 8.1 LP11 / 1986)
    Claims Claims 1-15 IF NOT
    The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).
     Opinion Base.-
    This opinion has been made on the basis of the patent application as published.
    State of the Art Report Page 3/4
     WRITTEN OPINION
    Application number: 201130576
     1. Documents considered.-
    The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.
    Document
    Publication or Identification Number publication date
    D01
    WO 2006101030 A1 (Q P CORP) 28.09.2006
    D02
    JP 2008179710 A (SHISEIDO CO LTD) 07.08.2008
  16.  2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement
    The present invention relates to a process for preparing a glycosaminoglycan with a methanol content of less than or equal to 10 ppm, comprising the following steps: a) arranging a glycosaminoglycan with a methanol content of more than 10 ppm in a container of equipment that it has a microwave and a vacuum pump incorporated; b) spray water on the glycosaminoglycan of stage a), under stirring, and c) subject the glycosaminoglycan of stage b) to microwave radiation, under vacuum and stirring, at a temperature below 70 ° C and for a period of time that allow to obtain glycosaminoglycan with a methanol content less than or equal to 10 ppm.
    The process allows to formulate said glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, in a product suitable for use in a food supplement or in a functional food.
    Document D01 describes a method of manufacturing low molecular weight hyaluronic acid and / or its salt, for use in food and cosmetic products. According to the invention, hyaluronic acid and / or its salt is dispersed in a medium containing an acidic solution of water. The medium can be ethanol, methanol and acetone. According to the preferred example of the invention, water containing 73% hydrochloric acid and ethanol is heated to 50 [degrees] C and an acid solution is prepared. The raw material containing fine sodium hyaluronate powder (6 kg) is dispersed in the medium. The mixture is heated to 50 [degrees] C and stirred continuously. The mixture is kept apart and the deposit is obtained by decantation. Subsequently, the preheated water containing hydrochloric acid and ethanol is added to the reservoir and stirred for 15 minutes. The method is repeated until the remaining hydrochloric acid is removed. The residue is dried under vacuum for 6 hours and low molecular weight hyaluronic acid is obtained.
    Document D02 describes a method for the production of low molecular weight hyaluronic acid at a low cost with a high yield without causing the discoloration of hyaluronic acid, which is suitable for mass industrial production. The hyaluronic acid powder is hydrolyzed in the presence of an acid by microwave irradiation in a low molecular weight substance in a state in which the hyaluronic acid powder is dispersed in a liquid medium containing water, so as not to dissolve the powder of hyaluronic acid. The medium containing the liquid medium water is a single-phase organic solvent that does not dissolve the hyaluronic acid powder but the water and an acid. The organic solvent is preferably selected from methanol, ethanol, propanol and isopropanol
    1.-NOVELTY None of the documents cited in the written opinion and in the state of the art report describes one that includes the combination of stages and conditions corresponding to the procedure of the patent application. Consequently, in the opinion of this Office, claims 1-15 comply with the requirement of novelty established in Art. 6.1 11/1986.
  17. 2.-INVENTIVE ACTIVITY
    As for the inventive activity, the application raises the problem of reducing the methanol content of glycosaminoglycans a. The solution is the procedure proposed in the invention that is developed under certain conditions: microwave under vacuum, water spray, stirring and temperature below 70 ° C. As a result, glycosaminoclycans with an amount of methanol less than or equal to 10 ppm are obtained.
    None of the documents cited, taken alone or in combination, clearly shows that such a procedure has the effect of the result obtained. Therefore, in the opinion of this Office, claims 1-15 comply with the inventive activity requirement established in Art. 8.1 11/1986.
    State of the Art Report Page 4/4
ES201130576A 2011-04-13 2011-04-13 Glycosaminoglycans with very low methanol content. Withdrawn - After Issue ES2390063B1 (en)

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* Cited by examiner, † Cited by third party
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US3895106A (en) 1970-04-15 1975-07-15 Morrison L M Novel CSA and CSC for use in man and mammals to inhibit atherosclerosis and the recurrence of cardiovascular incidents in atherosclerotic mammals
ES8800280A1 (en) * 1985-12-30 1987-10-16 Bioiberica Glycosamine-glycan prodn.
ES2223291B1 (en) 2003-08-06 2006-03-16 Bioiberica, S.A. New therapeutic use of condroitin sulfate.
ITMO20040199A1 (en) * 2004-07-29 2004-10-29 Biofer S P A Process for the purification from groups en no rsidui heparins of low molecular weight ottentite via nitrosation.
JP4576583B2 (en) * 2005-03-22 2010-11-10 キユーピー株式会社 Hyaluronic acid or a salt thereof, method for producing the same, and cosmetics and food compositions containing the same
JP5289936B2 (en) * 2006-02-24 2013-09-11 キユーピー株式会社 Novel low molecular weight hyaluronic acid and / or salt thereof, and cosmetics, pharmaceutical compositions and food compositions using the same
JP2008179710A (en) * 2007-01-25 2008-08-07 Shiseido Co Ltd Method for producing low-molecular-weight hyaluronic acid
US20080179318A1 (en) 2007-01-30 2008-07-31 Christopher John Cornwell Apparatus and Method for Vacuum Microwave Drying of Food Products
CL2007003320A1 (en) 2007-11-20 2008-04-25 Asesorias Y Servicios Solucion Palta dehydrated pieces; avocado process for dehydrating pieces comprising selecting avocado, precooling, scald, blunting, removing shell, applying antioxidant, slicing, apply antioxidant, salt, dehydrated by vacuum and microwave,
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