PROCESS FOR THE SEPARATION OF PINITOL FROM A CAROB EXTRACT

Abstract

A process is described for the separation of at least one inositol from a carob extract. The carob extract is filtered and demineralized, and has a Brix value greater than 60 and a pinitol content of 5 to 25 wt %. The carob extract is subjected to chromatographic separation which involves at least one passage on a chromatographic resin. This produces an aqueous solution comprising 35 to 70 wt % pinitol and a Brix value of 20 or lower. The aqueous solution is then purified to obtain a purified aqueous solution having a pinitol content of more than 55%.

Claims

1. A process for the separation of at least one inositol from a carob extract comprising: a) providing a filtered and demineralised carob extract having a Brix value of greater than 60 and a pinitol content, in weight percent based on the weight of the extract, from 5 to 25%; b) subjecting the carob extract to a chromatographic separation of the pinitol by at least one passage on a chromatographic resin to obtain an aqueous solution having a pinitol content, in weight percent based on the total weight of the solution, from 35 to 70%, and which has a Brix value of 20 or lower; and c) purifying the aqueous solution to obtain a purified aqueous solution having a pinitol content, in weight percent based on the total weight of the solution, of more than 55%.

2. The process of claim 1, wherein the carob extract of step a) is decoloured.

3. The process of claim 1, wherein the carob extract of step a) is demineralised by cationic exchange chromatography and anionic exchange chromatography.

4. The process of claim 3, wherein the carob extract of step a) is demineralised by passage of a carob extract on at least one anionic exchange resin and on at least one cationic exchange resin.

5. The process of claim 3, wherein the carob extract of step a) is demineralised by passage of a carob extract on at least two weak anionic exchange resins and on at least two strong cationic exchange resins.

6. The process of claim 5, wherein at least one of the passages of the carob extract on a weak anionic exchange resin is followed by the passage of the carob extract on a strong anionic exchange resin, before its passage on a strong cationic exchange resin.

7. The process of claim 3, wherein the demineralisation comprises subjecting a carob extract to the following steps in sequence: i. first passage of the carob extract on a weak anionic exchange resin; ii. first passage of the carob extract on a strong cationic exchange resin; iii. second passage of the carob extract on a weak anionic exchange resin; iv. passage of the carob extract on a strong anionic exchange resin; and v. second passage of the carob extract on a strong cationic exchange resin.

8. The process of claim 1, wherein the carob extract of step a) comprises, in weight percent based on the weight of the extract, from 5 to 20% of pinitol.

9. The process of claim 1, wherein step b) is carried out by means of a simulated moving bed chromatography technique.

10. The process of claim 1, wherein the purifying of step c) comprises concentrating the aqueous solution.

11. The process of claim 1, wherein the purified solution comprises pinitol at at least 70% purity.

12. The process of claim 1, further comprising: subjecting the aqueous solution to acid hydrolysis of the pinitol to obtain a solution comprising D-chiro-inositol, and subjecting the solution comprising D-chiro-inositol to chromatographic separation of the D-chiro-inositol by at least one passage on a strong anionic exchange resin to obtain an aqueous solution comprising D-chiro-inositol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG. 1 is a block diagram of a preferred embodiment of part of the process of the present invention, starting from carob pods until an aqueous solution is obtained with a pinitol content, in weight percent based on the total weight of the solution, from 35 to 70%, and having a Brix value of 20 or lower, of step b).

[0121] FIG. 2 shows the results of the HPLC analyses relating to the determination of the composition of the macerated and pressed carob extract described in Example 1.

[0122] FIG. 3 is a diagram of the passages relating to the demineralisation step according to a preferred embodiment of the invention (Example 1).

[0123] FIG. 4 shows the results of the HPLC analyses relating to the determination of the composition of the filtered, decoloured, rectified (demineralised) and concentrated carob extract described in Example 1.

[0124] FIG. 5 is a block diagram of a preferred embodiment of part of the process of the present invention, starting from the aqueous solution with a pinitol content, in weight percent based on the total weight of the solution, from 35 to 70%, and having a Brix value of 20 or lower, of step b) until the purified aqueous solution, of step c), is obtained (Example 1).

[0125] FIG. 6 shows the results of the HPLC analyses relating to the determination of the composition of the aqueous solution with a pinitol content, in weight percent based on the total weight of the solution, from to 70%, and having a Brix value of 20 or lower obtained in step b) described as a fraction 1 in Example 1.

[0126] FIG. 7 shows the results of the HPLC analyses relating to the determination of fraction 2 described in Example 1.

[0127] FIG. 8 shows the results of the HPLC analyses relating to the determination of the composition of the purified aqueous solution with a pinitol content, in weight percent based on the total weight of the solution, greater than 55% obtained in step c) described in Example 1.

[0128] FIG. 9 is a block diagram of a preferred embodiment of part of the process of the present invention, starting from the purified aqueous solution of step c), until the aqueous solution comprising D-chiro-inositol and having a Brix value of 1 or lower, of step d), is obtained. (Example 2).

[0129] FIG. 10 shows the results of the HPLC analyses relating to the determination of the composition of the aqueous solution comprising D-chiro-inositol and having a Brix value of 1 or lower, of step d), in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0130] The invention will now be further described with reference to embodiment examples provided for illustrative and non-limiting purposes.

Example 1

[0131] Process for the Separation of Pinitol (FIGS. 1-8)

[0132] 500 kg of carob pods were chopped until fragments of pods of about 1 cm in size were obtained and these fragments were macerated by mixing one part of the pods and three parts of water at 75° C. The fragments were then pressed, thus obtaining a carob extract having the following composition (in percent by dry weight on the dry weight of the juice): sucrose 62.5%; glucose 11.2%; pinitol 10.1%; fructose 16.1%; impurities 0.5% (composition in FIG. 2).

[0133] The aforesaid composition was determined by means of HPLC, eluent H.sub.2O, flow 0.6 ml/min, column temperature 75° C., column size 8 mmI.D, 300 mm column, functional group Ca, cationic exchange resin.

[0134] The obtained extract also had a Brix value of 18.

[0135] The extract was filtered with a rotary filter under vacuum using Perlite Randalite® W24 (Ceca Arkema Group, France) as a filter aid.

[0136] The filtrate was then subjected to a second filtration, with a bell filter, with filter elements arranged vertically, and having diatomaceous earth, in particular Dicalite Speedplus (Palumbo Trading Srl. Italy) as aid material.

[0137] The filtrate was then subjected to a third filtration, with the passage through the tangential filter, using membranes having a pore size of about 0.45 μm as filter elements.

[0138] The extract thus filtered was then passed on a Sepabeads SP207® (Resindion S.r.l., Italy) adsorbent resin for decolouration; and then subjected to demineralisation (or rectification) by means of passage on the following resins, in the described order (see diagram in FIG. 3):

[0139] 1) Column 1: Relite RAM1/M (Resindion S.r.l., Milan, IT) (weak anionic);

[0140] 2) Column 2: Relite RPS (Resindion S.r.l., Milan, IT) (strong cationic);

[0141] 3) Column 3: Relite RAM1/M (Resindion S.r.l., Milan, IT) (weak anionic);

[0142] 4) Column 3: Relite RAP1 (Resindion S.r.l., Milan, IT) (strong anionic); and

[0143] 5) Column 4: Relite RPS (Resindion S.r.l., Milan, IT) (strong cationic).

[0144] Table 1 shows the characteristics of each single resin.

TABLE-US-00001 TABLE 1 RESIN RESIN RESIN RESIN 2 RESIN 4 Strong 5 1 Anionic Cationic 3 Anionic Anionic Cationic RAM 1 RPS RAM 1 RAP 1 RPS Capacity 12000 14000 12000 4000 5000 Litres

[0145] Table 2 shows the operating conditions of each single column.

TABLE-US-00002 TABLE 2 RESIN RESIN RESIN RESIN RESIN 1 2 3 4 Strong 5 Anionic Cationic Anionic Anionic Cationic RAM 1 RPS RAM 1 RAP 1 RPS pH 11-6 1-8 11-6 11-6 2-6 Conductivity <4000 <4000 <500 <500 <150 μS/cm Total quantity 100 m.sup.3 of processed product

[0146] Table 3 shows the characteristics of the four resins mentioned above.

TABLE-US-00003 TABLE 3 Sepabeads Relite SP207 Relite RAP1 Relite RPS RAM1 (adsorbent (strong (strong (weak resin) anionic) cationic) anionic) Matrix Styrene-DVB Styrol-DVB Highly porous Highly copolymer porous styrol-DVB porous copolymer copolymer styrene-DVB copolymer Functional Trimethyl- Sulphonic Tertiary group amine amine Colour and Yellowish Light yellow Light brown Light yellow physical brown opaque opaque spheres opaque form spheres spherical beads Particle size 0.425- 0.425- 0.425- distribution 1.18 mm 1.18 mm 1.18 mm Ionic form Cl.sup.− N6.sup.+ Free base supplied Total 1.18 min eq/l 1.2 min eq/l 1.18 min eq/l 1.5 min eq/l exchange capacity Chemical Stability Stability Stability Stable stability within pH within pH within pH over the entire pH range Thermal 130° C. 60° C. max 120° C. max 100° C. stability (OH); 80° C. max max (Cl)

[0147] Table 4 shows the operating conditions of the four resins mentioned above.

TABLE-US-00004 TABLE 4 Sepabeads Relite Relite Relite SP207 RAP1 RPS RAM1 (adsorbent (strong (strong (weak resin) anionic) cationic) anionic) pH range 0-14 0-12 0-14 0-9 Operative 5-50 m/h 5-50 m/h 5-50 m/h linear flow rate Regenerant NaOH/Ethyl NaOH HCl NaOH Alcohol Regenerant 50-150 g/l 40-150 g/l 60-80 g/l level Displacement 1.5-2 BV 1.5-2 BV 1.5-2 BV 1.5-2 BV volume Washing 4-10 BV 4-10 BV 3-5 BV 4-8 BV volume

[0148] The extract therefore had conductivity values of 100 μS/cm, pH 3.10, and with regards to the colour a reading of 0.015 (reading Abs 430, optical path of the quartz cuvette 1 cm).

[0149] The extract thus obtained was then concentrated with heat, under vacuum conditions, passing from a temperature of 80° C. at the inlet to a temperature of 45° C. at the outlet, reaching 65° Bx. The extract had the following composition (in percent by dry weight on the dry weight of the juice): sucrose 5%; glucose 38%; pinitol 15%; fructose 38%; impurities 4% (see FIG. 4).

[0150] The aforesaid composition was determined, as described above, by means of HPLC, eluent H.sub.2O, flow 0.6 ml/min, column temperature 75° C., column size 8 mmI.D, 300 mm column, functional group Ca, cationic exchange resin. The concentrated extract thus obtained was then fed to an ISMB® plant (Improved Simulated Moving Bed, Mitsubishi Kasei Corporation) consisting of 4 UBK 530 columns (Resindion srl, Milan, Italy) and using demineralised water for elution.

[0151] Other operation parameters are summarised in Table 5.

TABLE-US-00005 TABLE 5 Resin volume 124 l Flow rate 59-60.6 l/h W/F 2.7 P/R 2.4 Temperature 60° C. Feed capacity 44-45 l/h Capacity of the pinitol fraction 16-17.8 l/h Key: W: water flow rate F: feed rate P: purified solution volume R: concentrate volume (“waste”)

[0152] Table 6 shows the ISBM operating conditions

TABLE-US-00006 TABLE 6 FLOW RATE SET Feed flow rate (Sop) (l/h) 16.39 Water flow rate (Wop) (l/h) 44.25 Purified solution flow rate (Fop) 42.8 (l/h) Concentrate flow rate (Gop) (l/h) 17.83 Recycle flow rate (R0op) (l/h) 44.25 VOLUME SET Feed volume (Qsop) (l) 2.6 Water volume (Qwop) (l) 7.13 Purified solution volume (Qfop) (l) 6.89 Concentrate volume (Qgop) (l) 2.87 Recycle volume (Q0op) (l) 16.43 DURATION OF THE STEPS (T1op) FEED TIME (sec) 580.04 (T0op) RECYCLING TIME (sec) 1336.62

[0153] From this chromatography two liquid fractions were obtained, having compositions summarised in Table 7 (see FIGS. 6 and 7, respectively).

TABLE-US-00007 TABLE 7 Fraction 1 Fraction 2 (purified) (waste) Brix 10 27 Sucrose 2.5 0 Glucose 21 7.3 Pinitol 70 6.3 Fructose 3 86

[0154] The aforesaid composition was determined by means of HPLC, as described above.

[0155] Fraction 1, containing 70% of pinitol, was then concentrated with heat under vacuum conditions, passing from a temperature of 80° C. at the inlet to a temperature of 45° C. at the outlet, until Brix values of 73 were obtained, and kept at 20° C. for 5 days, thus obtaining the formation of the crystals of pinitol.

[0156] After the formation and sedimentation of the crystal, 71% vol. ethyl alcohol was added to the concentrate in the proportion of two parts of alcohol and five parts of concentrate, to purify the crystal and obtain pinitol with a purity greater than 95%. The composition of the crystal obtained is as follows (in weight percent based on the weight of the concentrate): glucose 3%; pinitol 96.5%; sucrose 0%; fructose 0% (see FIG. 8).

[0157] The aforesaid composition was determined by means of HPLC under the conditions described above.

[0158] The purified solution was then subjected to centrifugation at 4000 rpm with the formation of a sediment containing pinitol and alcohol and a supernatant containing glucose and alcohol.

[0159] The sediment was subjected to dehumidification under heating, keeping it for two days at 45° C. thus obtaining 30 g of a white powder with a purity of the pinitol greater than 95%.

[0160] This result corresponds to a yield of 90% of pinitol by weight with respect to the weight of the pinitol present in the starting pods.

[0161] A comparison was then made between the white powder sample obtained and a standard pinitol sample, which confirmed the identity of the substance as pinitol.

[0162] For this purpose an aliquot of each sample was solubilised in a mixture of MeOH/H.sub.2O in an 80/20 ratio, in order to obtain a concentration of 15 ppm (μg/ml) for each sample.

[0163] The samples were analysed in LC/MS (liquid chromatography/mass spectrometry) using a Luna NH2 column (150×2.2, 3 μm). The analyses were conducted in isocratic elution using the mobile phase consisting of acetonitrile (80%) and water (20%). The analysis method lasts 15 minutes. The flow used is 300 μl/min.

[0164] Used instrumentation: Water Micromass Q-TOF Premier Mass Spectrometer.

[0165] The analysis confirmed the match between the two samples.

Example 2

[0166] Process for the Separation of the D-Chiro-Inositol (FIGS. 9, 10)

[0167] 30 g of powdered pinitol having a purity greater than 95% obtained in Example 1 were added to a 1-litre flask and introduced into 16 g of water and 104 g of 33% HCl were added to this solution.

[0168] The solution was heated for 20 minutes (from 45° C. to 60° C.) and 40 ml of 7.2 N HCl were added. At constant reflux, 50 ml of water were added. The solution was then brought to a boil and kept under boiling for 24 hours, during which the reflux remained constant.

[0169] After 24 hours, the solution was then subjected to decolouration by adding activated carbon to the solution (from 100 to 150 g/h) while keeping the solution under stirring for 60 minutes, thus obtaining 1160 ml of a solution having a Brix value of 6.5.

[0170] The solution was then subjected to filtration to eliminate the brown components formed during heating. The filtration with a rotary filter was carried out under vacuum using a mix at 50% by weight of Dicalite Speedplus® (Palumbo Trading, Srl, Italy) diatomaceous earth and at 50% by weight of perlite Randalite® W24 (Ceca Arkema Group, France) as an aid element.

[0171] The solution at this stage had a pH of 1, a clarity in NTU values (Nephelometric Turbidity Units) of 2, and was colourless.

[0172] The solution was then neutralised.

[0173] The solution was then subjected to passage on a strong anionic exchange resin (Relite RAP1) thus reaching a pH of 9-10 and then the solution was subjected to acidification with citric acid until a pH of 4.0 was reached.

[0174] The solution thus obtained had a Brix value of 0.3 and was then concentrated until a Brix value of 70 was reached.

[0175] The crystallisation of the D-chiro-inositol was then carried out keeping the solution at a temperature of 8° C. for 24 hours.

[0176] The concentrated solution had a D-chiro-inositol content of 95% or greater.

[0177] Finally, the concentrated solution was subjected to dehumidification with absorption thus obtaining 29 g of a white powder of D-chiro-inositol with a purity greater than 95%.

[0178] This result corresponds to almost 100% yield.

[0179] A comparison was then made between the white powder sample obtained and a standard D-chiro-inositol sample, which confirmed the identity of the substance as D-chiro-inositol, using the method described above in Example 1.

[0180] The analysis confirmed the match between the two samples.