阅读说明：本技术 从角豆提取物中分离松醇的方法 (Method for separating pinitol from carob bean extract ) 是由 F·S·迪塔 A·阿玛托 于 2020-04-30 设计创作，主要内容包括：描述了一种从角豆提取物中分离至少一种肌醇的方法,包括以下步骤：a)提供经过滤和脱矿质的角豆提取物,其白利糖度值大于60,松醇含量以提取物重量的重量百分比计为5％至25％；b)使步骤a)的所述角豆提取物进行松醇的色谱分离过程,其中所述过程包括使提取物在色谱树脂上通过至少一次,从而获得松醇含量以溶液总重量的重量百分比计为35％至70％的水溶液,且其白利糖度值为20或更低；和c)将步骤b)中如此获得的水溶液进行纯化步骤,从而获得松醇含量以溶液总重量的重量百分比计为大于55％的纯化水溶液。(A process is described for the isolation of at least one inositol from a carob bean extract comprising the steps of: a) providing a filtered and demineralized carob bean extract having a brix value greater than 60, a pinitol content of from 5% to 25% by weight of the extract; b) subjecting the carob bean extract of step a) to a process of chromatographic separation of pinitol, wherein said process comprises passing the extract at least once over a chromatographic resin, thereby obtaining an aqueous solution having a pinitol content of from 35% to 70% by weight, based on the total weight of the solution, and having a Brix value of 20 or less; and c) subjecting the aqueous solution thus obtained in step b) to a purification step, so as to obtain a purified aqueous solution having a pinitol content of greater than 55%, in percentages by weight of the total weight of the solution.)

1. A process for isolating at least one inositol from a carob bean extract comprising the steps of:

a) providing a filtered and demineralized carob bean extract having a brix value greater than 60, a pinitol content of from 5% to 25% by weight of the extract;

b) subjecting the carob bean extract of step a) to a process of chromatographic separation of pinitol, wherein said process comprises passing said extract at least once over a chromatographic resin, so as to obtain an aqueous solution having a pinitol content of from 35% to 70% by weight, relative to the total weight of the solution, and a Brix value of 20 or less; and

c) subjecting the aqueous solution thus obtained in step b) to a purification step, so as to obtain a purified aqueous solution having a pinitol content of more than 55%, in percentages by weight of the total weight of the solution.

2. The method of claim 1, wherein the carob bean extract of step a) is decolorized.

3. The process according to claim 1 or 2, wherein the carob bean extract of step a) is demineralized by cation exchange chromatography and anion exchange chromatography.

4. A process according to claim 3, wherein the carob bean extract of step a) is demineralized by passing the carob bean extract through at least one anion exchange resin and at least one cation exchange resin, more preferably a weak anion exchange resin and a strong cation exchange resin.

5. The process according to claim 3 or 4, wherein the carob bean extract of step a) is demineralized by passing the carob bean extract through at least two weak anion exchange resins and at least two strong cation exchange resins.

6. The method of claim 5, wherein after passing the carob seed extract at least once through a weak anion exchange resin, the carob seed extract is passed through a strong anion exchange resin and then through a strong cation exchange resin.

7. A process according to any one of claims 3 to 6, wherein the carob bean extract is demineralised sequentially by:

i. passing said carob seed extract through a weak anion exchange resin for a first time;

passing the carob bean extract over a strong cation exchange resin for a first time;

passing the carob tree extract a second time over a weak anion exchange resin;

passing the carob bean extract over a strong anion exchange resin; and

v. passing the carob bean extract a second time over a strong cation exchange resin.

8. The method according to any one of the preceding claims, wherein the carob bean extract of step a) comprises 5 to 20%, more preferably 10 to 15% pinitol as a weight percentage of the extract weight.

9. Process according to any one of the preceding claims, wherein step b) is carried out by a "simulated moving bed chromatography (SMB chromatography) technique, more preferably by a modified continuous chromatographic separation (ISMB), even more preferably(Mitsubishi Kasei Corporation).

10. The process according to any one of the preceding claims, wherein the purification step c) comprises a step of concentrating, preferably heating, the solution obtained in step b).

11. The process according to any one of the preceding claims, wherein at the end of purification step c) a concentrate is obtained comprising pinitol of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% purity.

12. The method according to any one of the preceding claims, wherein step c) is followed by step d): subjecting said aqueous solution obtained in step c) to acid hydrolysis of pinitol, thereby obtaining a solution containing D-chiro-inositol and subsequently chromatographically separating D-chiro-inositol from the solution comprising D-chiro-inositol by passing the aqueous solution comprising D-chiro-inositol at least once through a strong anion exchange resin, thereby obtaining an aqueous solution comprising D-chiro-inositol.

Technical Field

The present invention relates generally to the field of food supplement industry, in particular pinitol based food supplements.

In particular, the present invention relates to a method for isolating pinitol from a carob bean extract.

Background

Pinitol (3-O-methyl-1, 2, 4-cis-3, 5, 6-trans-hexahydroxycyclohexanol or 3-O-methyl-D-chiro-inositol) is D-chiro-inositol (C)₇H₁₄O₆) The molecular weight of the methyl ether (2) is 194.18 g/mol.

Pinitol (or D-pinitol) is known for its hypoglycemic action and its ability to improve insulin function when administered orally, as well as its use in the treatment of diabetes and obesity. Pinitol also increases creatine absorption in the same amount as carbohydrate intake. The effect allows the intake of a desired amount of creatine without the need to ingest large amounts of carbohydrate.

In addition, pinitol enhances the function of muscle tissue, increases the production of glycogen in muscle and stimulates the transport of glucose within muscle tissue. Said activity of pinitol can be used in the field of sports to improve the performance of athletes. In fact, pinitol has the effect of increasing glucose uptake by muscle cells and increasing storage of glycogen stored in muscle. This results in more stable blood glucose levels and higher energy levels, lasting longer over time.

Pinitol is administered orally in the form of a supplement or contained in food or drink, in a dose of 0.1mg to 1.0g per kg body weight per day. It may also be administered parenterally or intravenously.

Pinitol was first isolated from pine trees, but it was also present in soybeans at a concentration of about 1% (by weight of the dry weight of the soybeans). It is estimated that in some asian countries where soybean consumption is very common, pinitol intake by soybeans exceeds 5 mg/kg/day.

Pinitol is also present in plants of Bougainvillea spectabilis (Bougainvillea spectabilis) and syringa mexicana (glicidia sepium). Carob fruit (carob bean (Ceratonia siliqua)) also contains pinitol, and pinitol can be extracted from carob fruit by chromatography.

Carob is a long-life evergreen broad-leaved fruit tree, and grows slowly. In the food field, locust bean paste and seeds are used to produce chocolate substitutes, while many food thickeners and gelling agents are obtained from locust bean seed flour.

The carob bean extract generally has the following composition (in weight percent of the dry weight of the carob bean extract): 40-65% of sucrose; 7-15% of pinitol; 7-17% of fructose; 7-15% of glucose; 0.5 to 2 percent of impurity. Thus, carob is a very rich source of pinitol, higher than soy and pine needles (0.5-1% pinitol).

Patent EP 1241155B 1 (company General del algarroobo de Espana, SA) describes a process for the separation of pinitol from carob bean extracts, in which the sucrose contained in the extract is converted into fructose and glucose, and the syrup thus obtained is subjected to a chromatographic separation of pinitol from the sugars contained in the syrup, in particular by means of a strong cationic resin, so as to obtain an aqueous solution of pinitol having a purity of greater than 90%. Then, pinitol is separated from the solution.

Patent application KR 20040016338A (amigen co.ltd) describes a method for isolating pinitol from carob bean syrup, which comprises a step of culturing bacteria, yeasts or molds before isolation, in order to increase the pinitol content of the syrup and to obtain a product containing pinitol of low purity (40-50%). After the isolation of the microbial cells, the syrup thus obtained is subjected to an activated carbon treatment and a crystallization process. The result is a product containing pinitol of high purity, even greater than 90%.

Although both of the above-mentioned processes allow to obtain pinitol with a purity greater than 90%, they are very complex and expensive. In fact, in the case of patent EP 1241155, various filtration steps are envisaged, then first demineralization in a strong cationic resin (Na), then concentration of the extract, then on a cationic resinPerforming a sucrose inversion step, further demineralizing by anionic resin, and finally passingSeparating pinitol by chromatography. Each of these passes through a column requires the use of large amounts of water, as well as various concentration steps of the solution flowing from each column.

In the case of patent application KR20040016338, the step of culturing the microorganisms in the carob syrup requires a subsequent step of isolating the microbial cells. Both steps can be difficult on an industrial scale, also because they require special measures to manage microorganisms in the food field.

Pinitol can also be obtained by chemical synthesis, but this method is very expensive.

There is therefore a need in the art to provide a simpler and cheaper process for the separation of pinitol from carob beans (and subsequently of D-chiro-inositol starting from pinitol) than the processes of the prior art.

The technical problem underlying the present invention is therefore to provide a practical, inexpensive, versatile, scalable and high-yield process for the separation of pinitol (and D-chiro-inositol) from carob beans, in particular from carob bean extracts.

Summary of The Invention

According to the present invention, this problem has been solved by a process for the isolation of at least one inositol from a carob bean extract, comprising the steps of:

a) providing a filtered and demineralized carob bean extract having a Brix value (Brix value) greater than 60, a pinitol content ranging from 5% to 25% by weight of the extract;

b) subjecting the carob bean extract of step a) to a process of chromatographic separation of pinitol, wherein said process comprises passing the extract at least once over a chromatographic resin, thereby obtaining an aqueous solution having a pinitol content of from 35% to 70% by weight, based on the total weight of the solution, and having a Brix value of 20 or less; and

c) subjecting the aqueous solution thus obtained in step b) to a purification step, so as to obtain a purified aqueous solution having a pinitol content of more than 55%, in percentages by weight of the total weight of the solution.

The term "pinitol" in this patent refers to pinitol in the D configuration (D-pinitol), which is the only configuration of pinitol in the carob bean extract.

In this context, "carob extract" refers to an aqueous solution obtained by maceration and pressing of previously chopped carob pods and subsequent separation of the crude solid residue from the obtained aqueous solution.

Preferably, the at least one inositol is selected from pinitol and/or D-chiro-inositol, more preferably pinitol.

The impregnation is generally carried out by mixing the pods and water in a weight ratio of about 1:3 at a temperature of 60 ℃ to 90 ℃ for 1 to 24 hours, with a pH between 4.5 and 5.5. Pressing is usually carried out by means of a press, for example continuous pressing.

The resulting aqueous solution is generally darker in color and has suspended particles. Furthermore, the aqueous solution thus obtained is generally composed of glucose, fructose, sucrose, pinitol and other sugars or impurities and its brix value is generally from 10 to 30.

The filtered extract of step a) may be obtained by filtration techniques known in the art, preferably by a rotary vacuum filter, wherein more preferably the filter aid comprises perlite.

Preferably, the perlite has a particle size distribution of greater than 160 μm of between 5% (w/w) and 10% (w/w), more preferably 7% (w/w).

Preferably, the density value of the perlite is between 90 and 130g/l, more preferably 110 g/l.

Perlite suitable for the purposes of the invention are, for exampleW24(Ceca Arkema Group,France)。

Perlite consists of an alumino-silicate soft rock, which expands when heated. This expanded material is ground to form a filter aid of varying degrees.

Preferably, in addition to or as an alternative to filtration by means of a rotary filter, the filtration may comprise a filtration step by means of a bell filter, also known as a pre-coat filter, wherein the filter elements are arranged vertically.

Preferably, the filter material comprises diatomaceous earth.

Preferably, the diatomaceous earth comprises SiO₂。

Preferably, the diatomaceous earth is of flux calcined type (flux-calcined type).

Filter materials suitable for the purposes of the present invention are, for example, DicaliteOr Dicalite 6000(Palumbo tracing, Srl, Italy).

Preferably, the filtration may comprise a step of filtration by a tangential flow filter, according to techniques known in the art, in addition to or as an alternative to filtration by a rotary filter and/or filtration by a bell filter. Preferably, the tangential filter is equipped with a filter having a pore size of 0.45 μm or less.

Preferably, the carob bean extract of step a) is concentrated. The concentration is carried out by concentration techniques known in the art, such as concentration by heating, preferably at a temperature of 40 to 90 ℃ and a flow rate of 6000 to 10000 l/h.

Preferably, the carob bean extract of step a) is decolorized.

Preferably, the decolorization is performed by adsorption chromatography.

Preferably, the decolorization is carried out by passing the carob bean extract through an adsorbent resin, more preferably comprising a styrene-Divinylbenzene (DVB) copolymer based matrix. The selection of suitable resins and process parameters is within the skill of one in the art. Suitable resins for decolouration are adsorption resins of Resindion Srl (Milan Italy)SP207。

Preferably, the carob bean extract of step a) is demineralized (or refined) by cation exchange chromatography and anion exchange chromatography.

Preferably, the carob seed extract of step a) is demineralized (or refined) by passing the carob seed extract through at least one anion exchange resin and at least one cation exchange resin, more preferably a weak anion exchange resin and a strong cation exchange resin.

Preferably, the carob seed extract of step a) is demineralized by passing the carob seed extract sequentially through at least one anion exchange resin, more preferably a weak anion exchange resin, and subsequently through a cation exchange resin, more preferably a strong cation exchange resin.

Preferably, the carob seed extract of step a) is demineralized by passing the carob seed extract through at least two weak anion exchange resins and at least two strong cation exchange resins.

Preferably, the carob seed extract of step a) is demineralized by passing the carob seed extract through two weak anion exchange resins and two strong cation exchange resins.

Preferably, after passing the carob seed extract at least once through a weak anion exchange resin, the carob seed extract is passed through a strong anion exchange resin and then through a strong cation exchange resin.

Preferably, said passing of the carob seed extract at least once through the weak anion exchange resin is the last pass.

In a preferred embodiment, the carob bean extract is demineralized in the following sequence of steps:

i. passing the carob bean extract through a weak anion exchange resin for a first time;

passing the carob bean extract over a strong cation exchange resin for a first time;

passing the carob tree extract a second time over a weak anion exchange resin;

passing the carob bean extract over a strong anion exchange resin; and

v. passing the carob bean extract a second time over a strong cation exchange resin.

It is well known that strong ion exchange resins can function over the entire pH range of 0 to 12, whereas weak ion exchange resins can exchange only in a narrow range. Weak cation exchange resins function in the acidic range, while weak anion exchange resins function in the basic range.

Weak anion exchange resins suitable for use in the present invention include Relite(Resindion S.r.l.,Milan,IT)、MWA-1(Dow Chemical Company, JP) and Purolite^TM A100(Dow Chemical Company,JP)。

Strong anion exchange resins suitable for use in the present invention include Relite(Resindion S.r.l.,Milan,IT)、Amberlite^TMIRA900(Lenntech BV, NL) andA500(Lenntech BV,NL)。

strong cation exchange resins suitable for use in the present invention include Relite(Resindion S.r.l.,Milan,IT)、Amberlite^TMIRC200(Lenntech BV, NL) andA150(Lenntech BV,NL)。

preferably, the demineralization step is carried out continuously, i.e. without interrupting the demineralization process.

Preferably, the demineralization step eliminates 100% of the impurities and ions present in the extract.

Preferably, the pH of the solution leaving the demineralization step is between 3 and 5. In fact, in these pH ranges, browning of the extract can be avoided.

The selection of suitable resins and process parameters is within the skill of one in the art.

Preferably, the carob bean extract of step a) has a brix value of at least 65.

Brix (Brix: (B)^oBx) is a measure of the percentage of solid material (% w/w) dissolved in the liquid. In the present invention, the measurement of brix may be performed according to one of the methods known in the art, for example by means of a refractometer. A refractometer suitable for the purposes of the present invention is ATAGO RX-9000CX model (Atago USA, Inc., USA)

Preferably, the conductivity value of the extract of step a) is from 70 to 110. mu.S/cm, more preferably from 90 to 100. mu.S/cm. Conductivity measurements can be made according to methods known in the art, for example by a conductivity meter.

Preferably, the carob bean extract of step a) has a pH of 2 to 4.5, more preferably 2.5 to 3.5.

Preferably, the carob bean extract of step a) has an absorbance value of between 0.005 and 0.030, more preferably between 0.010 and 0.020, and has an optical path of 1cm and a wavelength of 430nm, read in a quartz cuvette.

Preferably, the carob bean extract of step a) comprises 5 to 20%, more preferably 10 to 15% pinitol, in weight percentages by weight of the extract.

Preferably, the carob bean extract of step a) comprises 5 to 15%, more preferably 8 to 10% sucrose, in weight percentages by weight of the extract weight.

Preferably, the carob bean extract of step a) comprises 5 to 15%, more preferably 8 to 10% sucrose; 5% to 20%, more preferably 10% to 15% pinitol; 20% to 50%, more preferably 30% to 40% fructose; 20% to 50%, more preferably 30% to 40% glucose, in weight percent of the extract weight.

Preferably, step b) is carried out by passing the carob bean extract of step a) through a strong cation exchange resin (Na +), such as the resin Diaion^TMUBK530(Resindion Srl, Milan, Italy). Another resin suitable for the purposes of the present invention is the resin Diaion^TMUBK535, UBK550 and UBK555(Resindion Srl, Milan, Italy).

Preferably, step b) is carried out by a (continuous) simulated moving bed chromatography (SMB chromatography) technique, more preferably by a modified (continuous) chromatographic separation ("modified simulated moving bed")"(ISMB)", e.g.(modified simulated moving bed, Mitsubishi Kasei Corporation).

Preferably, the simulated moving bed chromatography technique (SMB chromatography) described above is carried out using four chromatographic columns, preferably the continuous chromatography described above separates ISMB, in particular

As is well known, simulated moving bed chromatography is a continuous multi-column chromatography process, a technique which has been known since 1961 for the preparation of purified binary mixtures in a continuous manner.

The aforementioned ISMB technology was developed by mitsubishi chemical industries, inc (tokyo, japan), and is an improvement over the aforementioned SMB technology, allowing the separation of two components.

Preferably, in step b), elution is carried out with demineralized water.

Preferably, the aqueous solution obtained in step b) has a brix value of less than 15, more preferably 10 or less.

Preferably, the pinitol content of the above-mentioned aqueous solution obtained in step b) is comprised between 50 and 70%, more preferably between 60 and 70%, in weight percentages with respect to the total weight of the solution.

Preferably, the sucrose content of the above aqueous solution thus obtained in step b) is comprised between 2 and 8% in weight percentage with respect to the total weight of the solution.

Preferably, the above aqueous solution thus obtained in step b) has a sucrose content of 2 to 8%, a glucose content of 20 to 32%, a pinitol content of 50 to 70%, more preferably 60 to 70%, and a fructose content of 0 to 6%, in percentages by weight of the total weight of the solution.

Preferably, at the end of step b), a second (waste) solution is also obtained, having a brix value of between 25 and 40.

Preferably, the pinitol content of the second solution is 10% or less, as a weight percentage of the total weight of the solution.

Preferably, the second solution has a sucrose content of 0 to 4%, a glucose content of 2 to 10%, a pinitol content of 2 to 7%, and a fructose content of 85 to 95% in weight percent based on the total weight of the solution.

Preferably, the purification step c) comprises a concentration step, preferably concentration by heating, of the solution obtained in step b).

Preferably, the above solution heating concentration step comprises heating the solution to a temperature of 25 to 60 ℃ until the brix value reaches 60 or more, more preferably 70 or more, even more preferably 70 to 75.

Preferably, in step c), the crystallization step of the resulting solution is carried out after concentration of the solution.

Preferably, the crystallization step is carried out by holding the concentrated solution at a temperature of 18 to 25 ℃ for a period of 3 to 10 days until crystals are formed and precipitated.

Preferably, in step c), the crystallization is accomplished as follows: by adding ethanol (e.g. 71% by volume ethanol in water) to the concentrate thus obtained, and then precipitating the crystals until pure crystals are formed.

It is within the ability of the person skilled in the art to adjust the parameters and materials used in the purification step c) to obtain the desired results.

Preferably, at the end of the purification step c), a concentrate is obtained comprising pinitol of at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% purity.

In this patent, the purity of a component is understood to mean the weight percentage of the component based on the weight of the solution or crystal containing the component.

Preferably, the concentrate comprising pinitol having a purity of more than 55% discharged from step c) is centrifuged, thereby obtaining a precipitate comprising pinitol and a supernatant comprising glucose.

Preferably, the precipitate thus obtained is subjected to dehumidification under heating, more preferably at about 45 ℃ for at least two days, so as to obtain pinitol in the form of a white powder with a purity of at least 95%.

Preferably, step c) is followed by step d): subjecting the aqueous solution obtained in step c) or pinitol obtained after dehumidification of the pinitol precipitate to acid hydrolysis of pinitol, thereby obtaining a solution containing D-chiro-inositol, followed by chromatographic separation of D-chiro-inositol from the solution containing D-chiro-inositol at least once by passing the aqueous solution containing D-chiro-inositol through a strong anion exchange resin, thereby obtaining an aqueous solution containing D-chiro-inositol, preferably at least 95% by weight based on the total weight of the solution, and preferably having a Brix value of 1 or less.

Preferably, in step d), the acid hydrolysis is carried out by adding HCl, for example at 33% (v/v), to an aqueous solution of pinitol.

Preferably, the boiling step of the aqueous solution thus obtained is carried out for at least 12 hours, more preferably for at least 24 hours, after the addition of HCl.

Preferably, in step d), the strong anion exchange resin is selected from(Resindion Srl,Milan,IT)、Amberlite^TMIRA900(Lenntech BV, NL) anda500(Lenntech BV, NL), preferably

Preferably, in step d), the aqueous solution is subjected to a decolorization step before being passed through the strong anion exchange resin, more preferably by adding activated carbon to the solution.

Preferably, the activated carbon is added to the solution in a concentration of 50 to 150 grams per hundred liters of solution, more preferably 80 to 120 grams per hundred liters of solution.

The activated carbon is preferably selected from activated carbons having a median diameter of from 4 to 50 μm, more preferably from 8 to 15 μm.

Preferably, the BET of the activated carbon is between 1200 and 2000m²A value between/g, more preferably between 1500 and 1800m²Between/g.

Adapted for the purposes of the inventionThe activated carbon is, for example, Picapure HP 120(Pica Italia SpA, Italy) or(Italy)。

The median diameter (MT50 or d50) is understood to be the diameter measured by means of a laser particle sizer, which corresponds to 50% by weight of particles having a smaller diameter and 50% by weight of particles having a higher weight. Diameter refers to the particle size as measured by a laser particle sizer as previously described.

The BET surface area is intended to be measured by the ASTM D-3037/89 protocol.

Preferably, in step d), the aqueous solution entering the strong anion exchange resin has a brix value of 6.5 or more.

Preferably, in step d), the aqueous solution leaving the strong anion exchange resin has a basic pH, more preferably between 8 and 12.

Preferably, the aqueous solution comprising D-chiro-inositol obtained in step D) (leaving the strong anion exchange resin) is acidified, thereby obtaining an acidified aqueous solution having a pH between 3 and 5, more preferably about 4.

Preferably, the acidification step is carried out with a weak acid, such as citric acid.

Preferably, the aqueous solution comprising D-chiro-inositol obtained in step D) is concentrated to obtain a concentrated aqueous solution having a brix value of 60 or more, more preferably 65 or more, even more preferably 70 or more.

Preferably, the concentrated aqueous solution thus obtained is subjected to crystallization, more preferably the aqueous solution is kept at a temperature of about 7-10 ℃ for 2-6 hours.

Preferably, after crystallization, D-chiro-inositol is subjected to dehumidification, more preferably absorption dehumidification, to obtain D-chiro-inositol with a purity of at least 90%, more preferably at least 95%.

Preferably, the yield of pinitol is at least 3%, more preferably at least 5%, even more preferably at least 7%, most preferably from 7 to 10%, in weight percentage with respect to the weight of the pods from which it is extracted.

Preferably, the yield of pinitol is 15% or less, in weight percentage with respect to the weight of the pods from which it is extracted.

Preferably, the yield of pinitol is 80% or higher, in weight percentage with respect to the weight of the starting pinitol (present in the pods).

Preferably, the process of the invention is carried out continuously.

Thus, the process of the present invention involves the separation of pinitol, D-chiro-inositol, or both. The process can in fact be carried out until step c) to obtain pinitol or it can be continued to obtain D-chiro-inositol starting from pinitol. It is also possible to obtain D-chiro-inositol by using only a part of pinitol, thereby obtaining both pinitol and D-chiro-inositol.

It was surprisingly found that, thanks to the process of the present invention, pinitol and/or D-chiro-inositol of high purity can be prepared in a simpler, faster and cheaper way than the processes of the prior art.

In fact, the process of the invention envisages a relatively small number of passes (passes) relative to the prior art.

Furthermore, due to the presence of the demineralization step, especially when the demineralization step is performed according to the preferred embodiment of the present invention, the separation of pinitol can be performed starting from a relatively highly concentrated glycidyl solution having a relatively highly concentrated pinitol. This powerful feature makes the process more elaborate, since the amounts of aqueous solution involved are relatively small (equal in pinitol content). Furthermore, the amount of water required for dilution is relatively small in each chromatography pass (pass), thereby reducing costs and waste.

In a preferred embodiment, the resin sequence employed, in particular the preferred sequence i to v, is particularly advantageous in the demineralization step, which comprises in succession i) a weakly anionic resin, ii) a strongly cationic resin, iii) a weakly anionic resin, iv) a strongly anionic resin, and finally v) a strongly cationic resin.

The alternation of anion and cation resins, and the alternation of strong ion exchange resins and weak ion exchange resins, makes the recovery of pinitol particularly high. It is also particularly advantageous if the last column pass is carried out on a strong cationic resin, since this results in the effluent solution having an acidic pH, thereby avoiding browning of the solution and thus avoiding the need to carry out a special decolourisation step.

Another advantage of the process of the invention is that it can be carried out continuously. The process of the invention results in simpler, higher automation and process speed compared to batch processes.

Brief description of the drawings

Fig. 1 is a block diagram of a preferred embodiment of a part of the process of the invention, starting from the pods until an aqueous solution is obtained having a pinitol content of 35 to 70%, and a brix value of 20 or less, in weight percentages based on the total weight of the solution, of step b).

FIG. 2 shows the results of HPLC analysis in connection with determining the composition of the extract of macerated and crushed carob beans as described in example 1.

Figure 3 is a passage diagram relating to a demineralization step according to a preferred embodiment (example 1) of the present invention.

FIG. 4 shows the results of HPLC analysis in connection with determining the composition of filtered, bleached, refined (demineralized) and concentrated carob extracts as described in example 1.

Fig. 5 is a block diagram of a preferred embodiment of a part of the process of the invention, starting from the aqueous solution of step b) having a pinitol content of 35 to 70% by weight, based on the total weight of the solution, and a brix value of 20 or less, until the purified aqueous solution of step c) is obtained (example 1).

Fig. 6 shows the results of HPLC analysis related to the determination of the composition of the aqueous solution obtained in step b) described as fraction (fraction)1 in example 1, having a pinitol content of 35 to 70% in weight percentage of the total weight of the solution, and a brix value of 20 or less.

Figure 7 shows the HPLC analysis results associated with the determination of fraction 2 as described in example 1.

Figure 8 shows the results of HPLC analysis related to the determination of the composition of the purified aqueous solution obtained in step c) described in example 1, having a pinitol content greater than 55%, in percentages by weight of the total weight of the solution.

FIG. 9 is a block diagram of a preferred embodiment of a part of the process of the invention, starting from the purified aqueous solution of step c) until an aqueous solution comprising D-chiro-inositol and having a Brix value of 1 or less of step D) is obtained. (example 2).

FIG. 10 shows the results of HPLC analysis relating to determination of the composition of an aqueous solution containing D-chiro-inositol and having a Brix value of 1 or less of step D) in example 2.

Detailed Description

The invention will now be further described with reference to embodiments provided for illustrative and non-limiting purposes.

Example 1

Separation process of pinitol (FIGS. 1-8)

500kg of pods were chopped until approximately 1cm pieces of pods were obtained and these pieces were impregnated by mixing one part of pods with three parts of water at 75 ℃. The splits were then pressed, obtaining a carob bean extract with the following composition (in dry weight percentage of the dry weight of the juice): 62.5 percent of sucrose; 11.2% of glucose; 10.1% of pinitol; 16.1 percent of fructose; 0.5% impurity (composition in fig. 2).

The above composition was determined by HPLC and eluent H₂O, flow rate of 0.6ml/min, column temperature of 75 ℃, column size of 8mmI.D, 300mm column, functional group Ca and cation exchange resin.

The resulting extract had a brix value of 18.

Using PerliteW24(Ceca Arkema Group, France) was used as a filter aid and the extract was filtered under vacuum on a rotary filter.

The filtrate was then filtered a second time with a bell filter, the filter elements being arranged vertically and diatomaceous earth, in particular Dicalite speed plus (Palumbo tracking srl. italy), as auxiliary material.

The filtrate was then subjected to a third filtration through a tangential flow filter using a membrane having a pore size of about 0.45 μm as the filter element.

The extract thus filtered is then passed through Sepabeads(resolution s.r.l., Italy) adsorbent resin for decolorization; demineralization (or refining) was then carried out by the following resins in the order described (see the diagram in figure 3):

1) column 1: relite RAM1/M (resolution S.r.l., Milan, IT) (weak anion);

2) column 2: reite RPS (resolution s.r.l., milan, IT) (strong cation);

3) column 3: relite RAM1/M (resolution S.r.l., Milan, IT) (weak anion);

4) column 4: reite RAP1 (resolution s.r.l., milan, IT) (strong anion); and

5) column 5: reite RPS (resolution s.r.l., milan, IT) (strong cation).

Table 1 shows the properties of each resin.

TABLE 1

Table 2 shows the operating conditions for each column.

TABLE 2

Table 3 shows the characteristics of the above four resins.

TABLE 3

Table 4 shows the working conditions of the four resins described above.

TABLE 4

Thus, the conductivity value of the extract was 100. mu.S/cm, the pH was 3.10, and the color read was 0.015 (reading Abs 430, optical path of the quartz cuvette was 1 cm).

The extract thus obtained was then concentrated by heating under vacuum from an inlet temperature of 80 ℃ to an outlet temperature of 45 ℃ to 65 ℃ Bx. The extract had the following composition (in dry weight percent of the dry weight of the juice): 5% of sucrose; 38% of glucose; 15% of pinitol; 38% of fructose; impurity 4% (see fig. 4).

The above composition was determined by HPLC as described above, eluent H₂O, flow rate of 0.6ml/min, column temperature of 75 ℃, column size of 8mm I.D, 300mm column, functional group Ca, cation exchange resin.

The concentrated extract thus obtained is then fed to a column consisting of 4 UBK530 columns (Resindion srl, Milan, Italy)The apparatus (modified simulated moving bed, Mitsubishi Kasei Corporation) and elution were performed using demineralized water.

Other operating parameters are summarized in table 5.

TABLE 5

Volume of resin	124l
		Flow rate of flow	59-60.6l/h
W/F	2.7
		P/R	2.4
Temperature of	60℃
		Feed capacity	44-45l/h
Volume of pinitol fraction	16-17.8l/h

Note:

w: velocity of water flow

F: feed rate

P: volume of purified liquid

R: volume of concentrate ("waste")

Table 6 shows the ISBM operating conditions

TABLE 6

Two liquid fractions were obtained from the chromatogram, the compositions of which are summarized in table 7 (see fig. 6 and 7, respectively).

TABLE 7

	Fraction 1 (purification)	Fraction 2 (waste)
			Brix degree	10	27
Sucrose	2.5	0
			Glucose	21	7.3
Pinitol	70	6.3
			Fructose	3	86

The above composition was determined by HPLC as described above.

Fraction 1 containing 70% pinitol is then concentrated under vacuum with heating from an inlet temperature of 80 ℃ to an outlet temperature of 45 ℃ until a brix value of 73 is obtained and left at 20 ℃ for 5 days, thereby forming pinitol crystals.

After crystal formation and precipitation, 71% by volume of ethanol was added to the concentrate in a ratio of two parts ethanol to five parts concentrate to purify the crystals and obtain pinitol with a purity of greater than 95%. The composition of the crystals obtained was as follows (in weight percent based on the weight of the concentrate): 3% of glucose; pinitol 96.5%; 0% of sucrose; fructose 0% (see fig. 8).

The above composition was determined by HPLC under the above conditions.

The purified solution was then centrifuged at 4000rpm to form a precipitate containing pinitol and ethanol and a supernatant containing glucose and ethanol.

The precipitate was dehumidified under heating and kept at 45 ℃ for two days, thereby obtaining 30g of pinitol white powder having a purity of more than 95%.

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

Then, a comparison was made between the obtained white powder sample and a standard pinitol sample, and it was confirmed that the substance was pinitol.

To this end, an aliquot of each sample was dissolved in MeOH/H in a ratio of 80/20₂O mixture to obtain a concentration of 15ppm (μ g/ml) for each sample.

Using Luna NH₂The column (150X 2.2, 3 μm) was used to analyze the samples by LC/MS (liquid chromatography/mass spectrometry). Isocratic elution analysis was performed using a mobile phase consisting of acetonitrile (80%) and water (20%). The analytical method lasted 15 minutes. The flow rate used was 300. mu.l/min.

The apparatus used was: water Micromass Q-TOF Premier mass spectrometer.

Analysis confirmed the identity between the two samples.

Example 2

Method for separating D-chiro-inositol (FIGS. 9 and 10)

30g of the powdery pinitol having a purity of more than 95% obtained in example 1 was put into a 1 liter flask and 16g of water was added, and 104g of 33% HCl was added to the solution.

The solution was heated for 20 minutes (from 45 ℃ C. to 60 ℃ C.) and 40ml of 7.2N HCl was added. At constant reflux, 50 ml of water were added. The solution was then boiled and kept boiling for 24 hours, during which time the reflux was kept constant.

After 24 hours, the solution was decolorized by adding activated carbon (100 to 150g/h) to the solution while keeping the solution stirred for 60 minutes, thereby obtaining 1160 ml of a solution having a brix value of 6.5.

The solution was then filtered to remove the brown component formed during heating. Using 50% by weight of Dicalite(Palumbo tracing, Srl, Italy) diatomaceous earth and 50% perliteA mixture of W24(Ceca Arkema Group, France) was used as an auxiliary material and filtered under vacuum with a rotary filter.

The pH of the solution at this stage was 1, the NTU value (nephelometric turbidity units) was 2 for clarity (clarity) and was colorless.

The solution is then neutralized.

The solution was then passed through a strong anion exchange resin (Relite RAP1) to bring the pH to 9-10, and then the solution was acidified with citric acid until the pH reached 4.0.

The solution thus obtained had a brix value of 0.3 and was then concentrated until a brix value of 70 was reached.

Then, D-chiro-inositol was crystallized and the solution was kept at a temperature of 8 ℃ for 24 hours.

The concentrate has a D-chiro-inositol content of 95% or more.

Finally, the concentrated solution is absorbed and dehumidified, and 29g of D-chiro-inositol white powder with the purity of more than 95 percent is obtained.

This result corresponds to a yield of almost 100%.

Then, a comparison was made between the obtained white powder sample and a standard D-chiro-inositol sample, and the substance was confirmed to be D-chiro-inositol using the method described in example 1 above.

Analysis confirmed the identity between the two samples.