The present invention relates to a process for preparing a glycosaminoglycan with a methanol content less than or equal to 10 ppm. The process allows formulating 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 high molecular weight polymeric biomolecules consisting of a repeated dimeric structure. They are essentially found in living organisms where they carry out different physiological functions. Among the glycosaminoglycans, chondroitin sulfate, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate and keratan sulfate should be highlighted. Except for hyaluronic acid, all the rest contain sulfate groups in their structures.

Chondroitin sulfate has a polymer structure characterized by a repeating disaccharide made up of /V-acetyl-D-galactosamine and D-glucuronic acid. Most of the /V-acetyl-D-galactosamine residues are sulfated. It generally has a molecular weight between 10,000 and 70,000 Da, depending on the origin and method for obtaining it (N. Volpi, Journal of Pharmaceutical Sciences, 96(12), 3168-3180, (2007)). The usefulness of chondroitin sulfate in treating cardiovascular diseases (US 3,895,106), psoriasis (WO2005/014012) or arthrosis (M.G. Lequesne, Rev. Rhum. Eng. Ed., 61, 69-73 (1994); G. Verbruggen et ai, Osteoarthritis Cart., 6 (Supplement A), 37-38 (1998)) has been described.

Hyaluronic acid is a non sulfated glycosaminoglycan with a polymer structure characterized by a repeating disaccharide made up of /V-acetyl-D- glucosamine and D-glucuronic acid monosaccharides. It is one of the main components of cartilage, synovial membrane and synovial fluid. Its use in treating arthrosis, generally intraarticularly, is particularly significant. Its use in ophthalmology for speeding up wound healing, as well as in cosmetics, has also been described. Chondroitin sulfate, hyaluronic acid, dermatan sulfate and keratan sulfate glycosaminoglycans are used both in nutrition, in the form of food supplements or functional foods, as well as in pharmaceutical preparations.

Methanol is used as the solvent in many process for preparing chondroitin sulfate, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate and keratan sulfate glycosaminoglycans from animal tissues (trachea, mucosa, comb, etc.) since using non-denatured ethanol is very expensive. The amount of residual methanol of the glycosaminoglycans obtained through some of the process described in the literature is approximately 50-2,000 ppm.

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

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

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

Microwaves are used in different industrial sectors, such as in the food, ceramic, rubber, paper, and pharmaceutical sectors.

Microwave vacuum drying is the result of combining two technologies: microwaves which provide a fast and uniform drying, allowing that the heat is uniformly generated inside the solid and the vacuum which allows reducing the drying temperatures. It is widely used in the food processing industry, for example, for dehydrating fruits and vegetables (see, for example, patent application WO2009/066259; patent application US2008/0179318) and in the pharmaceutical industry, for example, for drying and/or granulating 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 -2146 (2005); CM. McLoughlin et al., Drying Technology, 21 (9), 1719-1733 (2003)) or for dehydrating temperature-sensitive biological materials (patent application WO2010/124375).

In view of the foregoing, finding a process for preparing glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, with a methanol content less than or equal to 10 ppm is of great interest, so that it can be used for preparing a functional food or food supplement, meeting the requirements of the competent authorities.

A process for preparing glycosaminoglycan, for example chondroitin sulfate or hyaluronic acid, having an amount of methanol less than or equal to 10 ppm from a glycosaminoglycan obtained by means of a process in which methanol has been used as the solvent has not been described to date.

Disclosure of the Invention

The present inventors have surprisingly found that the process of the present invention allows preparing a glycosaminoglycan, preferably chondroitin sulfate or hyaluronic acid, with a methanol content less than or equal to 10 ppm. In the present invention, a glycosaminoglycan comprising an amount of methanol less than or equal to 10 ppm is surprisingly obtained when water is sprayed under stirring on a glycosaminoglycan containing an amount of methanol greater than 10 ppm and it is subjected to microwaving under vacuum. Therefore, the glycosaminoglycan thus obtained can be used in preparing food supplements or functional foods.

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

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

b) spraying 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 less than 70°C and for a time period which allows obtaining the glycosaminoglycan with a methanol content less than or equal to 10 ppm. To control the temperature during this step, water can be circulated through the outer jacket of the container containing the g lycosam i n og lyca n .

In a preferred embodiment, the process comprises an additional step before step b), in which the glycosaminoglycan of step a) is subjected to microwave radiation, under vacuum and stirring, at a temperature less than 70°C and for a time period which allows obtaining a glycosaminoglycan with a methanol content comprised between 10 ppm and 150 ppm, preferably of 100 ppm or 88 ppm. To control the temperature during this additional step, water can be circulated through the outer jacket of the container containing the g lycosam i n og lyca n .

In another equally preferred embodiment, when the temperature reaches 70°C in step 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 the vacuum are connected again. This cycle of steps (i), (ii), (iii), (iv) and (v) can be repeated several times until reaching a methanol content less than or equal to 10 ppm.

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 the vacuum are connected again. This cycle of steps (i), (ii), (iii) and (iv) can be repeated several times until reaching a methanol content less than or equal to 10 ppm.

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 it is comprised between 151 ppm and 2,000 ppm, more preferably it is 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 glycosaminoglycan of step a), preferably at 40 or 42 watts per kg of glycosaminoglycan of step 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 sprayed is comprised between 1 % and 3% by weight of water with respect to the weight of the glycosaminoglycan of step a), preferably the amount of water sprayed is 1 .5% by weight of water with respect to the weight of the glycosaminoglycan of step a).

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, keratan sulfate and mixtures thereof. The glycosaminoglycan is preferably chondroitin sulfate or hyaluronic acid.

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

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

The glycosaminoglycans used in the process of the present invention as starting materials can be obtained from different animal tissues, such as for example, rooster combs, porcine trachea, bovine trachea, porcine mucosa, bovine mucosa, skin, bird sternum or skeleton of elasmobranch fish, such as sharks, through the processes described in the literature, using methanol or denatured ethanol with methanol as a solvent. For example, the chondroitin sulfate can be obtained by means of proteolytic enzymatic digestion of cartilaginous animal tissues, for example cattle or pig tracheas. The hyaluronic acid can be obtained from rooster combs, which, once ground up, are digested with a proteolytic enzyme. The enzyme is subsequently deactivated by means of heating, it is 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 the starting glycosaminoglycans used in the present invention contain an amount of methanol greater than 10 ppm.

The chondroitin sulfate from cartilaginous tissue is mainly found in two isomeric forms, differing in the position of the sulfate group present in the N- acetylgalactosamine, chondroitin 4-sulfate (chondroitin sulfate A; R ¹ =S0 ₃ ^" Na ⁺ , R ² =H) and chondroitin 6-sulfate (chondroitin sulfate C; R ¹ =H, R ² = S0 ₃ ^" Na ⁺ ) residue, which are represented by the following structure:

R ¹ = S0 ₃ Na ⁺ , R ² = H

R ² = S0 ₃ Na ⁺ , R ¹ = H

The chondroitin sulfate can also contain sulfate groups in positions 4 and 6 of the /V-acetylgalactosamine residue (chondroitin sulfate E), or in position 6 of /V-acetylgalactosamine and position 2 of the D-glucuronic acid (chondroitin sulfate D).

Hyaluronic acid is a non sulfated glycosaminoglycan having a molecular weight comprised between 100,000 daltons and 3,000,000 daltons. Its polymer structure is characterized by a repeating disaccharide made up of /V-acetyl-D- glucosamine and D-glucuronic acid:

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

When "ppm" is mentioned in the present invention, it refers to "parts per million". It is a concentration measurement unit and refers to the number of units of substance, methanol in the present invention, per every million units of the mixture, the starting glycosaminoglycan in the present invention. The word "spray" of the present invention has the same meaning as the word "pulverize".

When "rpm" is mentioned in the present invention, it refers to "revolutions per minute".

When "under stirring" is mentioned in the present invention, it refers to a stirring which allows homogenizing the glycosaminoglycan, especially when water is sprayed.

When "impeller" is mentioned in the present invention, it refers to an impeller-mixer blade which rotates, stirring the glycosaminoglycan.

When "chopper" is mentioned in the present invention, it refers to a system of knifes rotating at high speed, stirring 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 for housing the glycosaminoglycan which is capable of receiving the microwave radiation produced by the generator, a vacuum system capable of applying a vacuum to the chamber, an outer jacket of the container containing the glycosaminoglycan through which water or another medium for maintaining the glycosaminoglycan at the desired temperature can circulate, stirring means, for example an impeller and a chopper which can be introduced in the glycosaminoglycan for carrying out good stirring, and water spraying means can be used. For example, a commercial mixer-granulator, such as the Collette Ultima-Pro 25 L or the Lbb Bohle VMA 300 mixer-granulator can be used.

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

The impeller speed can be, for example, 50 rpm and the chopper speed can be, for example, 600 rpm in the step in which it is irradiated without spraying water.

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

The vacuum pump of the equipment 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 sprayed on the glycosaminoglycan can be ion- and microorganism-free water, and it can be sprayed with a peristaltic pump.

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

A glycosaminoglycan, for example chondroitin sulfate or hyaluronic acid, with a methanol content less than or equal to 10 ppm is not obtained from the processes described in the literature which use methanol or denatured ethanol with methanol as a solvent. The chondroitin sulfate obtained in the processes in which methanol or denatured ethanol with methanol is used as a solvent generally 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 with microwaves under vacuum without spraying water, the methanol content is not reduced below 45 ppm; in contrast, if water is sprayed under stirring prior to microwaving under vacuum, 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 proper stirring, part of the product dissolves forming lumps.

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

The present inventors have found that the methanol content of a glycosaminoglycan drops below 10 ppm or down to 10 ppm with determined conditions: microwaving under vacuum, spraying water, stirring and a temperature less than 70°C.

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

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

Brief Description of the Drawings

Figure 1 depicts the ppm of methanol over time in minutes, when a chondroitin sulfate is subjected to microwave radiation under vacuum with or without spraying water. It is seen starting from 375 minutes.

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

Detailed Description of the Preferred Embodiments

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 means of microwavinq under vacuum and spraying water

10 kg of chondroitin sulfate containing 88 ppm of methanol were introduced in the container of an equipment having microwaves at a frequency of 2,450 MHz (Collette Ultima-Pro 25 L mixer-granulator) which incorporated a vacuum pump. The chondroitin sulfate was stirred by means of the impeller (at 200 rpm) and the chopper (at 2,500 rpm) of the equipment, maintaining the chondroitin sulfate at a temperature less than 70°C by means of an outer jacket of the container through which water circulated. Subsequently, and always under stirring, 150 g of water (1.5% by weight of water with respect to the weight of the starting chondroitin sulfate) were sprayed, vacuum was created until reaching 50 mbar and it was irradiated with microwaves at a power of 40 watts per kg of the starting chondroitin sulfate (400 watts in total). When the temperature reached 70°C, the microwave was stopped, the vacuum was broken, the chondroitin sulfate was cooled by means of the water circulating through the outer jacket of the container containing it, 150 g of water were again sprayed, vacuum was created until 50 mbar and it was irradiated with microwaves at a power of 40 watts per kg of the starting chondroitin sulfate (400 watts in total). This cycle with steps of stopping the microwave, breaking the vacuum, cooling the chondroitin sulfate, spraying water, creating the vacuum and irradiating with microwave was repeated every time the temperature reached 70°C. After the cycle or cycles, the microwave was stopped, the vacuum was broken and chondroitin sulfate with a methanol content of 8.3 ppm was recovered.

The ppm of methanol were 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 means of microwavinq under vacuum and spraying water

1 12.5 kg of chondroitin sulfate with a methanol content of 198 ppm were weighed and placed inside the container of an equipment having microwaves at a frequency of 2,450 MHz (Lbb Bohle VMA 300 mixer-granulator) which incorporated a vacuum pump. The chondroitin sulfate was stirred by means of the impeller (at 50 rpm) and the chopper (at 600 rpm) of the equipment, maintaining the chondroitin sulfate at a temperature less than 70°C by means of an outer jacket of the container through which water at 20°C circulated. Vacuum was then created until reaching 70 mbar and it was irradiated with microwaves 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 impeller speed was increased (to 220 rpm) and the chopper speed was increased (to 1 ,500 rpm), maintaining the chondroitin sulfate at a temperature less than 70°C by means of the jacket outside the container through which water circulated. Subsequently, and always under stirring, 1 .68 kg of water (1 .5% by weight of water with respect to the weight of the starting chondroitin sulfate) were sprayed, vacuum was created until reaching 70 mbar and it was irradiated with microwaves for 2 hours at a power of 42 watts per kg of the starting chondroitin sulfate. After this time, the microwave was stopped and the vacuum was broken. 1 .68 kg of water were then sprayed always under stirring, vacuum was created until reaching 70 mbar and the microwave was connected, irradiating for 2 hours at a power of 42 watts per kg of starting chondroitin sulfate. In total six cycles were carried out with the following consecutive steps per cycle: stopping the microwave, breaking the vacuum, spraying water, creating the vacuum and irradiating for 2 hours. After the six cycles the microwave was stopped, the vacuum was broken and chondroitin sulfate with a methanol content of 6.4 ppm was recovered.

This example demonstrates that the process can be applied for preparing large amounts of chondroitin sulfate with a methanol content less than 10 ppm.

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

The present example was carried out for studying the effect of irradiating with microwaves under vacuum without spraying water for the purpose of comparing 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 an equipment having microwaves at a frequency of 2,450 MHz (Collette Ultima-Pro 25 L mixer-granulator) which incorporated a vacuum pump. The chondroitin sulfate was stirred by means of the impeller (at 50 rpm) and the chopper (at 600 rpm) of the equipment, maintaining the chondroitin sulfate at a temperature less than 70°C by means of an outer jacket of the container through which water at 20°C circulated. The vacuum was then created until reaching 50 mbar and it was irradiated with microwaves for 8 hours at a power of 40 watts per kg of starting chondroitin sulfate (400 watts in total). The temperature of the chondroitin sulfate contained in the container of the equipment was kept under control throughout the entire process so that it did not exceed 70°C. After this time the microwave was stopped, the vacuum was broken and chondroitin sulfate with a methanol content of 88 ppm was recovered.

Example 4: Study of the elimination of methanol from a chondroitin sulfate sample by means of microwaving under vacuum. Effect of spraying water in the elimination of methanol

The process of Example 3 was followed, but starting from 7 kg of chondroitin sulfate containing 588 ppm of methanol, and stopping the process several times for collecting samples. Chondroitin sulfate with a methanol content of 91.7 ppm was obtained. In Figure 1 the process is seen starting from 375 minutes. As it can be observed, at 442 minutes a value of 98.7 ppm of methanol was reached. After irradiating for another 26 minutes, it only dropped down to a value of 91 .7 ppm of methanol.

Then, starting from the previous chondroitin sulfate containing 91 .7 ppm of methanol, the process of Example 1 was applied, namely, water was sprayed and it was irradiated with microwaves under vacuum. As it can be observed in Figure 1 , at 524 minutes (56 minutes with the conditions of Example 1 ) the methanol content dropped to 33.3 ppm, and at 1 ,019 minutes (551 minutes with the conditions of Example 1 ) 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 containing less amount of methanol, specifically containing 43.8 ppm of methanol. After applying the process of Example 3 (without spraying water), the amount of methanol was reduced very little, because at 269 minutes it only dropped to 41.3 ppm (see Figure 2).

Then, starting from the previous chondroitin sulfate having 41.3 ppm of methanol, the process of Example 1 was applied, namely, water was sprayed and it was irradiated with microwaves under vacuum. As it can be observed in Figure 2, at 358 minutes (89 minutes with the conditions of Example 1 ) the methanol content dropped to 26.9 ppm, and at 606 minutes (337 minutes with the conditions of Example 1 ) the methanol content dropped to 10.8 ppm.

Taking into account these results, it can be concluded that in order to eliminate methanol from a glycosaminoglycan, in this case from a chondroitin sulfate, to an amount less than or equal to 10 ppm, spraying water during the process of irradiation with microwaves under vacuum is essential.