Method of recovering pinitol or chiro-inositol in high yield from soy fractions Patent Application (2025)

U.S. patent application number 10/297043 was filed with the patent office on 2003-10-02 for method of recovering pinitol or chiro-inositol in high yield from soy fractions. Invention is credited to Choi, Chi-Man, Jeon, Yeong-Joong, Kim, Jong-Jin, Shin, Yong-Chul.

Method of recovering pinitol or chiro-inositol in high yield fromsoy fractions

Abstract

This invention relates to a method for recovering usefulproducts from soy fractions with high efficiency, and morespecifically to increasing the recovery yield of pinitol orchiro-inositol from soy fractions, in which it is contained by aprocess comprising the steps of culturing a microorganism totransform pinitol derivatives into pinitol in soy fractions,thereby to increase the pinitol content in soy fractions, followedby removing microorganisms, insoluble materials and othermacromolecules from said fractions by centrifugation or filtrationto obtain an aqueous solution containing pinitol or chiro-inositol,contacting said solution with activated carbon to adsorb thepinitol or chiro-inositol, contacting said solution with activatedcarbon to adsorb the pinitol or chiro-inositol, and then recoveringit by stepwise or gradient elution with an organic solvent.

 David A Einhorn Anderson Kill & Olick 1251 Avenue of the Americas New York NY 10020 US

Claims

1. A method for recovering chiro-inositol ingredients from soyfractions, in which the soy fractions are provided in a liquidphase and the microbes are cultured in the soy fractions toincrease the content of pinitol or chiro-inositol, said microbesbeing selected from the group consisting of bacteria, yeasts,fungi, or combinations thereof.

2. The method as set forth in claim 1, wherein said microbes belongto the genus Saccharomyces.

3. The method as set forth in claim 1, wherein said microbes belongto the genus Aspergillus.

4. The method as set forth in claim 2, wherein said microbes areSaccharomyces calsbergensis.

5. The method as set forth in claim 3, wherein said microbes areAspergillus niger.

6. A method for isolating chiro-inositol ingredients from soyfractions, in which the soy fractions are provided in a liquidphase and passed through activated charcoal to adsorb thechiro-inositol ingredients and other saccharides onto the activatedcharcoal.

7. The method as set forth in claim 6, wherein the chiro-inositolingredients comprise chiro-inositol and pinitol.

8. The method as set forth in claim 6, wherein the chiro-inositolingredients are eluated with an aqueous solution of an organicsolvent.

9. The method as set forth in claim 8, wherein the organic solventis selected from the group consisting of ethanol, isopropanol,methanol and acetone.

10. The method as set forth in claim 8, wherein the chiro-inositolingredients are eluted separately from other saccharides byincreasing the concentration of the aqueous solution stepwise orcontinuously.

11. The method as set forth in claim 10, wherein the aqueoussolution has a concentration of 1-20% in the early elution stageand 20-100% in the final elution stage.

12. A method for isolating chiro-inositol ingredients from soyfractions, comprising the steps of: providing the soy fractions asa liquid phase sample; removing insoluble matters andmacromolecules from the liquid phase sample by centrifugation orfiltration; passing the liquid phase sample removed of insolublematters and macromolecules through a column packed with activatedcharcoal to adsorb the chiro-inositol ingredients onto theactivated charcoal; washing the column with distilled water toremove molecules remaining unadsorbed; and eluting the adsorbatechiro-inositol ingredients with a stepwise or continuousconcentration gradient of an aqueous organic solution, said aqueousorganic solution ranging in concentration from 5 to 20% (v/v) andbeing selected from the group consisting of solutions of methanol,ethanol, isopropanol, and acetone in water.

13. A method for isolating chiro-inositol ingredients from soyfractions, comprising the steps of: providing the soy fractions asa liquid phase sample; culturing at least one microbial species inthe liquid phase sample to increase the content of pinitol orchiro-inositol therein; removing the microbial mass generatedduring culturing, insoluble matters and macromolecules from theculture by centrifugation or filtration; and recovering pinitol orchiro-inositol from the supernatant or filtrate by activatedcharcoal column chromatography or ion exchange chromatography.

14. The method as set forth in claim 13, wherein the recovery stepis carried out by the method claimed in any of claims 7 to 12.

15. The method as set forth in any one of claims 6 to 11, furthercomprising the step of recovering useful ingredients other thanchiro-inositol ingredients from the soy fractions, said usefulingredients comprising isoflavone or soybean oligosaccharides.

16. A method for isolating chiro-inositol ingredients from soyfractions, comprising the steps of: providing the soy fractions ina liquid phase and concentrating them; culturing at least onemicrobial species in the concentrate to increase the content ofpinitol or chiro-inositol therein, said microbial species beingselected from the group consisting of bacteria, yeasts, and fungi;concentrating the culture to a solid content of 50-70% (w/w) andadding the concentrate with a 95% ethanol solution in an amount aslarge as one to three volumes of the remaining liquid portion ofthe concentrate to further precipitate insoluble matters; removingthe solid content by centrifugation or filtration; vaporizing theethanol contained in the supernatant or filtrate, said supernatantor filterate being enriched in pinitol; passing the supernatant orfiltrate deprived of ethanol through an activated charcoal columnto adsorb pinitol or chiro-inositol onto the activated charcoal andeluting the adsorbate with an eluent; and concentrating the eluateand crystallizing chiro-inositol or pinitol.

17. The method as set forth in claim 1, 6 or 12, wherein the soyfractions are selected from the group belonging to the followingcategories: (i) soybean, or defatted soybean meal or its extracts,(ii) soybean curd whey (iii) soy molasses (iv) concentrates of (i)or (ii).

18. The method as set forth in claim 1, 6, 12, 13 or 16, whereinthe chiro-inositol ingredients comprise chiro-inositol and pinitol.(iii) soy molasses (iv) concentrates of (i) or (ii).

19. The method as set forth in claim 1, 6, 12, 13 or 6, wherein thechiro-inositol ingredients comprise chiro-inositol and pinitol.

Description

TECHNICAL FIELD

[0001] The present invention relates, in general, to the recoveryof pinitol or chiro-inositol from soy fractions and, particularly,to a method of isolating pinitol or chiro-inositol at highefficiency with economic benefit from soybean curd whey, which iswasted after the production of soybean curd from soybean; or fromsoy molasses left after production of soy proteins; or fromhydrothermal solution of defatted soybean meals, by microbialtreatment and activated charcoal column chromatography.

BACKGROUND ART

[0002] With recent increasing interest in health, various attemptshave been made to develop novel health food materials. Consideredas a health food material, soybean is now studied for its novelfunctions in the body.

[0003] In addition to being highly valuable in a sitologicalaspect, soybean is known to have a variety of physiologicalactivities, including activities against cancer, arteriosclerosis,oxidation and bacteria, and blood glucose reduction. Responsiblefor such physiological activities, ingredients of soybean areexemplified by isoflavone, saponin, lecithin, trypsin inhibitor,etc. Isoflavone, which was proven effective in preventing cancerand osteoporosis, has been commercialized as a material for healthfoods in many advanced countries. Also, soybean oligosaccharides,such as raffinose and stachyose, were identified as being effectivefor promoting growth of beneficial intestinal bacteria, andcommercialized in Japan.

[0004] In expectation of the existence of other biologically activematerials in soybean, much active research has been conducted. As aresult, soybean was found to contain chiro-inositol and its methylether derivative, called pinitol. Recently, these sugars haveattracted particular attention since finding that they are usefulin reducing serum glucose in type 2 (insulin-independent)diabetics.

[0005] As well known, chiro-inositol is an epimer of myo-inositol,and pinitol has a methyl group linked to the carbon at position 3via an ether bond.

[0006] Since the early 1990s, the serum glucose-reducing effect ofchiro-inositol has been verified in many reports (Ortmeyer et al.,Endocrinol. 651, 1993; Huang et al., Endocrinol. 132:652-657, 1993;Farese et al., Proc. Natl. Acad. Sci. USA, 91:11040-11044, 1994;Fonteles et al., Diabetologia 39;731-734,1996). This naturallyoccurring compound is found to show no side effects, such asgastroenteric or hepatic troubles that conventional oralhypoglycemics have, and does not cause hypoglycemia even uponoveruse. With this safety advantage, chiro-inositol has highpossibility to be successfully developed as health food materialsor medicines. Besides, chiro-inositol is suggested as beingtherapeutically effective for the treatment of obesity andpolycystic ovarian syndrome (Nestler J. E. et al., New Eng. J.Med., 340:1314-1320, 1999). Pinitol, which is predominant overother chiro-inocitol derivatives in soybean, is also found to showthe same hypoglycemic effect as that of chiro-inositol (U.S. Pat.No. 5,827,896; Narayanan et al., Current Science, 56(3):139-141,1987).

[0007] Diverse methods have been suggested for preparingchiro-inositol, thus far. For example, hydrolysis of pinitol(methyl ether of D-chiro-inositol) extracted from plant leaves(Anderson et al., Ind. Eng. Chem., 45:593-596, 1953), andorganochemical conversion of myo-inositol to chiro-inositol (Shenet al., Tetrahedron Letters, 131:1105-1108, 1990) are reported.However, these prior art methods are economically unfavorablebecause they take a long time and show low yield in the preparationof chiro-inositol. In addition, chiro-inositol can be synthesizedfrom kasugamycin (U.S. Pat. No. 5,091,596). However, this method islow in efficiency, so that chiro-inositol is produced at highcost.

[0008] Aiming to more efficient production of chiro-inositol, thepresent inventors have researched the use of edible resources richin chiro-inositol, such as soybean, its processed foodstuffs, andpine needles, as materials for reducing serum glucose levels(Korean Pat. Appl'n No. 10-2000-12881) and isolation andpurification of chiro-inositol from such edible resources by acidhydrolysis (Korean Pat. Appl'n No. 10-2000-12882). Also, thepresent inventors developed the use of activated charcoal column inseparating pinitol and chiro-inositol (Korean Pat. Appl'n No.10-2001-001611), which is economically favorable in comparison withprior art methods, such as use of zeolite (U.S. Pat. No.4,482,761), cation exchange resins (U.S. Pat. No. 5,096,594) andanion exchange resins (U.S. Pat. No. 5,482,631). In addition, thepresent inventors made a study of effective recovery of pinitol andchiro-inositol (Korean Pat. Appl'n No. 10-2001-44677), in whichmicroorganisms such as bacteria, yeasts, and fungi are used toconvert precursors existing as glycosides or phosphorus compoundsto pinitol and chiro-inositol.

DISCLOSURE OF THE INVENTION

[0009] Leading to the present invention, the intensive and thoroughresearch into the economic production of pinitol orchiro-inositol(hereinafter both referred generally to as"chiro-inositol ingredients"), conducted by the present inventors,resulted in the finding that soy fractions contain chiro-inositolingredients and are enriched in chiro-inositol ingredient contentby treatment with certain microbes, and chromatography eluting withan organic solvent such as ethanol through a column packed withactivated charcoal allows the chiro-inositol ingredients to beisolated with a purity of 90% or higher.

[0010] Therefore, it is an object of the present invention toprovide a method for recovering chiro-inositol ingredients from soyfractions at high efficiency.

[0011] It is another object of the present invention to provide arecovery method of chiro-inositol ingredients, which iseconomically favorable.

[0012] It is a further object of the present invention to provide amethod for recovering chiro-inositol ingredients and other usefulingredients from soy fractions, with ease.

[0013] In accordance with an aspect of the present invention, thereis provided a method for recovering chiro-inositol ingredients fromsoy fractions, in which the soy fractions are provided in a liquidphase and the microbes are cultured in the soy fractions toincrease the content of pinitol or chiro-inositol, said microbesbeing selected from the group consisting of bacteria, yeasts,fungi, or combinations thereof.

[0014] In accordance with another aspect of the present invention,there is provided a method for isolating chiro-inositol ingredientsfrom soy fractions, comprising the steps of: providing the soyfractions as a liquid phase sample; removing insoluble matters andmacromolecules from the liquid phase sample by centrifugation orfiltration; passing the liquid phase sample removed of insolublematters and macromolecules through a column packed with activatedcharcoal to adsorb the chiro-inositol ingredients onto theactivated charcoal; washing the column with distilled water toremove molecules remaining unadsorbed; and eluting the adsorbatechiro-inositol ingredients with a stepwise or continuousconcentration gradient of an aqueous organic solution, said aqueousorganic solution ranging in concentration from 5 to 20% (v/v) andbeing selected from the group consisting of solutions of methanol,ethanol, isopropanol, and acetone in water.

[0015] In accordance with a further aspect of the presentinvention, there is provided a method for isolating chiro-inositolingredients from soy fractions, comprising the steps of: providingthe soy fractions as a liquid phase sample; culturing at least onemicrobial species in the liquid phase sample to increase thecontent of pinitol or chiro-inositol therein; removing themicrobial mass generated during culturing, insoluble matters andmacromolecules from the culture by centrifugation or filtration;and recovering pinitol or chiro-inositol from the supernatant orfiltrate by activated charcoal column chromatography or ionexchange chromatography.

[0016] In accordance with still a further aspect of the presentinvention, there is provided a method for isolating chiro-inositolingredients from soy fractions, comprising the steps of: providingthe soy fractions in a liquid phase and concentrating them;culturing at least one microbial species in the concentrate toincrease the content of pinitol or chiro-inositol therein, saidmicrobial species being selected from the group consisting ofbacteria, yeasts, and fungi; concentrating the culture to a solidcontent of 50-70% (w/w) and adding the concentrate with a 95%ethanol solution in an amount as large as one to three volumes ofthe remaining liquid portion of the concentrate to furtherprecipitate insoluble matters; removing the solid content bycentrifugation or filtration; vaporizing the ethanol contained inthe supernatant or filtrate, said supernatant or filtrate beingenriched in pinitol; passing the supernatant or filtrate deprivedof ethanol through an activated charcoal column to adsorb pinitolor chiro-inositol onto the activated charcoal and eluting theadsorbate with an eluent; and concentrating the eluate andcrystallizing chiro-inositol or pinitol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and other advantagesof the present invention will be more clearly understood from thefollowing detailed description taken in conjunction with theaccompanying drawings, in which:

[0018] FIG. 1 is a process flow illustrating the isolation ofpinitol and chiro-inositol from soy fractions by use of anactivated charcoal column;

[0019] FIG. 2 is a chromatogram obtained by column chromatographyeluting pinitol (PI), chiro-inositol (CI) and oligosaccharides (OS)from a column of a size of 500 ml with 500 ml of 10% (v/v) ethanoland 500 ml of 50% (v/v) ethanol at a rate of 500 m/min;

[0020] FIG. 3 is a chromatogram obtained by column chromatographyeluting pinitol (PI) and oligosaccharides (OS) from a column of asize of 10 L with 10 L of 10% (v/v) ethanol and 10 L of 50% (v/v)ethanol at a rate of 10 L/min;

[0021] FIG. 4 is a process flow associated with microbialtreatment, illustrating the isolation of pinitol and chiro-inositolfrom soy fractions by use of an activated charcoal column; and

[0022] FIG. 5 is a process flow illustrating the production ofpinitol of high purity according to a preferred embodiment of thepresent invention.

BEST MODES FOR CARRYING OUT THE INVENTION

[0023] In general, the present invention is directed to therecovery of pinitol and/or chiro-inositol at high yield fromnatural resources, such as fruits of Glycine soja S. et Z., GlycineMax(L.) Merr., soybean, bean leaves, bean buds, defatted soybean,bean chaff, bean sprouts, pine needles, pine bud, inner layer ofpine bark, etc.

[0024] In the present invention, microbes such as bacteria, yeasts,or fungi are cultured in edible resources containing pinitol orchiro-inositol, e.g., soy fractions, to increase the content ofpinitol or chiro-inositol and these compounds are selectivelyadsorbed and eluted by use of a column packed with activatedcharcoal, whereby pinitol or chiro-inositol can be produced at highyield with economic benefit. Quite different from prior arts, thepresent invention can remove saccharides from soy fractions tobring about a significant reduction in the load of organicscontained in the final waste, as well as recovering chiro-inositolingredients in a small volume with a high concentration, thusisolating chiro-inositol ingredients at high efficiency.

[0025] The term "soy fractions" as used herein means soybean curdwhey, which is generally wasted after the production of soybeancurd, soy molasses left after the production of soybean proteins, ahydrothermal solution of defatted soybean meal, or mixturesthereof.

[0026] Generally, 1 g of soybean contains 3.4-6.8 mg ofchiro-inositol ingredients that are found to exist aschiro-inositol at an amount of 15-20%, as pinitol at an amount of25-35% and as chiro-inositol or pinitol glycosides or phosphoruscompounds at an amount of 50-60%. Soybean curd whey also containsvarious chiro-inositol ingredients: 15-20% in the form ofchiro-inositol; 30-40% in the form of pinitol; and the remainding50-60% in the form of glycosides or phosphorous compounds ofchiro-inositol or pinitol (in detail, pinitol glycosides, pinitolphosphates, pinitol phytates, pinitol phospholipids, pinitolesters, lipid-bound pinitol). Likewise, other soy fractions containvarious chiro-inositol compounds in which glycosides or phosphorouscompounds of pinitol exist in significant amounts, in spite of thepredominance of pinitol itself. Different from pinitol in physicalproperties, such pinitol derivatives cannot be recovered byconventional pinitol recovery processes.

[0027] Leading to the present invention, the intensive and thoroughresearch into the recovery of pinitol or chiro-inositol, conductedby the present inventors, resulted in the finding that, as timegoes by after the generation of soybean curd whey, thechiro-inositol ingredient composition of the soybean curd wheyshifts toward increased free chiro-inositol and pinitol, with nomodulation in the total amount of chiro-inositol ingredients in thesoybean curd whey. It was also found that the conversion ofderivatives of chiro-inositol and pinitol into free chiro-inositoland pinitol, useful for reducing serum glucose levels, is owed tothe biological action of the microbes living in the soybean whey,such as bacteria, yeasts, fungi, etc. In the present invention,pinitol derivatives contained in soy fractions can be convertedinto free pinitol through treatment with such microbes, therebygreatly improving the recovery of pinitol from soy fractions. Inaddition, other saccharides can be removed from the soy fractions,thereby brining about a significant reduction in the burden ofsubsequent recovery processes as well as in the load of organicscontained in the final waste.

[0028] If they convert pinitol derivatives into free pinitol andare harmless, bacteria, yeasts and fungi all can be used in thepresent invention. Of course, the kind of the microbe useddetermines conversion conditions, recovery efficiencies of pinitol,and removal rates of other saccharides. Among the microbes,Sacharomyces calsbergensis is of special interest for the presentinventors because it is found to have the highest ability toproduce pinitol as well as to remove sugars. After being used toincrease the content of chiro-inositol ingredients, microbes areremoved from, for example, soybean curd whey and recovered invarious subsequent processes.

[0029] In the present invention, dominant species were selectedfrom among the microbes that naturally live in soybean curd wheyand increase the pinitol content, and measured for pinitolproduction ability. The results are given in Table 1, below. Tothis end, soybean curd whey was autoclaved at 121.degree. C. for 15min and used as a medium in which two naturally separated yeastspecies and three bacterial species were cultured, alone or incombination, for 5 days with observation of the change in pinitoland chiro-inositol content.

1TABLE 1 Pinitol Production Ability of Microbes Separated fromSoybean Curd Whey Strain Pinitol (mg/L) Chiro-Inositol (mg/L)Intact Whey 467 65 Mixed Microbes 616 129 Yeast Y1 614 156 Y2 619166 Bacteria B1 525 128 B2 625 142 B3 608 136

[0030] As apparent from the data of Table 1, all of the microbes,irrespective of their kinds, make a contribution to the increase ofpinitol and chiro-inositol content. The same experiments performedwith other microbes led to the conclusion that the increase of thepinitol content in soybean curd whey is made by all microbes ratherthan specific microbes. However, pinitol increase rates differ fromone microbe species to another. In the assimilation ability of thesaccharides contained in soybean curd whey, there is alsodifference among microbe species. To complete the presentinvention, there were selected microbes that satisfy the followingconditions: 1) rapid pinitol production, 2) efficient removal ofsaccharides from whey, and 3) safety for food processing. Theresults are given in Table 2, below.

2TABLE 2 Pinitol Production of Various Microbes from Soybean CurdWhey Pinitol Production Sugar Strain Rate assimilation Use.sup.1Yeast S. carlsbergensis +++++ +++++ Beer S. cerevisae ++++ ++invertase S. pastorianus +++ ++ Beer C. utilities +++ + 5-adenylicacid Fungi A. niger ++++ +++++ .alpha.-amylase P. funiculosum ++ ++pectinase T. viride ++++ + xylanase Bacteria B. stearothermophillus+++ + .alpha.-amylase E. coli ++++ + -- P. amyloderamosa ++ +isoamylase .sup.1according to Japanese Food AdditiveRegulations

[0031] As seen in Table 2, the microbes having superb pinitolproduction rates can be exemplified by Saccharomyces calsbergensis,Saccharomyces cerevisiae, Aspergillus niger, Trichoderma viride,and E. coli with preference for Saccaromyces calsbergensis. Insaccharide assimilation ability, Saccharomyces calsbergensis andAspergillus niger are superior to the other microbes. Meanwhile,fungi including Aspergillus niger were observed to increase thepinitol content to day 2 or 3 of culturing, but decrease it afterthat. It is believed that fungi utilize the produced pinitol as anutrient in the late culture stage owing to the shortage of othernutrients. The microbes summarized in Table 2 are regarded as safefor food processing because they acquired permission for at leastone use according to Japanese Food Additive Regulations. Takentogether, the data obtained in the experiments demonstrate thatSaccharomyces calsbergensis is the most preferable for producingpinitol.

[0032] Pinitol production was also conducted using defatted soybeanmeal. In this regard, defatted soybean meal is powdered anddissolved in hot water and the powder remaining unsolved wasremoved. Then, the hydrothermal solution was treated withSaccharomyces calsbergensis. The final pinitol content was measuredto be increased by 1.5-2.0 fold, compared to the initial content,as with soybean curd whey. In the case that the microbe wasinoculated in the hydrothermal solution which was not removed ofthe undissolved defatted soybean meal powder, the final pinitolcontent was increased to 2.0-2.5 times the initial content. This isbelieved to be attributed to the fact that glycosides present inthe powder are extracted to the hydrothermal solution duringculturing.

[0033] An experiment was executed to determine whether theconcentration increase of pinitol is attributed to certain enzymesproduced from microbes. Saccharomyces calsbergensis and Aspergillusniger were cultured in soybean curd whey and filtered off through a0.45.mu. filter. The soybean curd whey thus free of microorganismswas combined with another sterile soybean curd whey and incubatedfor two days. No increase was observed in pinitol concentration,nor the composition changes. From this fact, it can be inferredthat the concentration increase of pinitol by microbes results fromcomplex biological mechanisms of microbes, rather than actions ofone or two enzymes. For use in inoculation with microbes, thesoybean curd whey or defatted soybean meal may be concentrated by afactor of 10-20 fold.

[0034] In accordance with the present invention, the recovery ofchiro-inositol ingredients from soybean curd whey, soy molasses, orhydrothermal extracts of defatted soybean meal resorts to the useof activated charcoal columns. Soybean curd whey or hydrothermalextracts of defatted soybean meals contain large quantities ofoligosaccharides as well as pinitol and chiro-inositol. In thepresent invention, pinitol and chiro-inositol are separated fromother saccharides by use of an activated charcoal column. Pinitoland chiro-inositol are adsorbed, together with other saccharides,onto activated charcoal, but can be eluted separately from othersaccharides with a concentration gradient of an organic eluent suchas ethanol.

[0035] Below, a detailed description will be given of the recoveryof chiro-inositol ingredients from soy fractions by use of anactivated charcoal column in conjunction with FIG. 1.

[0036] In accordance with an aspect of the present invention, thereis provided a method for recovering chiro-inositol ingredients fromsoy fraction using an activated charcoal column, which is brokendown into the pretreatment process of removing insolubleingredients and macromolecules such as proteins from soy fractions,the adsorption process of binding molecules of interest ontoactivated charcoal, the elution process of detaching the moleculesfrom activated charcoal, and the post-treatment process ofrecovering chiro-inositol ingredients as powder. In thepretreatment process, insoluble matters or polymeric materials areremoved by centrifugation or filtered off. The filtered sample isloaded onto a column filled with activated charcoal andchiro-inositol ingredients of the sample are adsorbed ontoactivated charcoal during passage through the column. Afteringredients remaining unadsorbed are washed off with distilledwater, the adsorbates are eluted with an organic solvent, such asmethanol, ethanol, isopropanol or acetone. This eluent is fed at acontinuous or stepwise concentration gradient from 5 to 20% (v/v).The activated charcoal is recycled by washing with distilled water.The eluate containing chiro-inositol is concentrated to a desiredpurity. More details are described as follows.

[0037] First Process: Pretreatment

[0038] In this pretreatment process, two tasks are performed inorder to better the efficiency of the activated charcoal columnprocess, a core process in the present invention. First, insolublematters are removed from soy fractions with the aid of a centrifugeor filter. Insoluble matters, unless removed, clog the activatedcharcoal column to impede its normal absorption action. The othertask is to remove macromolecules such as proteins from soyfractions by ultrafiltration. Once they are adsorbed onto activatedcharcoal, proteinaceous matters are hardly detached therefrom,thereby significantly decreasing the lifetime of the activecolumn.

[0039] Second Process: Absorption into Activated Charcoal-FilledColumn

[0040] In this process, a sample deprived of insoluble matters andproteinaceous matters in the pretreatment process is passed throughan activated charcoal-filled column, during which chiro-inositolingredients and soybean oligosaccharides are adsorbed onto theactivated charcoal. To increase the absorption capacity of theactivated charcoal, it is adjusted to the pH range of 6 to 8 withcaustic soda. In one round, the column filled with thepH-controlled activated charcoal can take care of a soybean curdwhey with a total solid content of 25 g chiro-inositol in an amountof as large as 5-10 volumes thereof. Preferably, the soybean curdwhey is fed at a rate of 1-2 bed volumes (hereinbefore referred asto "BV") per hour.

[0041] Third Process: Elution from Activated Charcoal Column

[0042] To remove matters that remain unadsorbed onto the activatedcharcoal, the column is washed with 1-2 BV of distilled water.Appropriate washing rates of the activated charcoal column fallinto the range of 1-2 BV per hour. An eluent containing 5-20% (v/v)organic solvent is fed in an amount of 1-2 BV to elute from theactivated charcoal chiro-inositol alone. Examples of the usefulorganic solvents include methanol, ethanol, isopropanol and acetonewith highest preference for ethanol in consideration ofworkability, safety, and economic benefit. The eluent is preferablyin the pH range of 3 to 4 at which chiro-inositol ingredients havelow partition coefficients, and is fed at a rate of 0.5-2 BV perhour into the column. Afterwards, a regeneration solutioncontaining the same solvent at a concentration of 40-80% is passedin an amount of 1 BV through the column to remove the mattersadsorbed strongly onto the activated charcoal. Also, theregeneration solution is preferably fed at a rate of 0.5-1 BV perhour.

[0043] The eluate from the column is collected in fraction andanalyzed by high performance liquid chromatography (HPLC), followedby pooling the fractions found to contain chiro-inositolingredients. Those who have sufficient experience in this type ofelution can harvest the fractions rich in chiro-inositol only bymonitoring the changes of solvent concentration with the aid of arefractometer without additional fractionation work.

[0044] Under such eluting conditions, the adsorbates are eluted inthe order of increasing adsorptive force. For example, pinitolfractions first come out of the column at 0.6-2.2 BV, thenchiro-inositol fractions at 2.0-3.0 BV, and finally oligosaccharidefractions at 2.4-4.0 BV. In the pinitol fractions, the totalcontent of chiro-inositol ingredients amounts to 55-70%, which isbroken down into 50-60% of pinitol and 2-10% of chiro-inositol. Thechiro-inositol fractions contain 5-10% of pinitol and 10-15% ofchiro-inositol. Also, the chiro-inositol fractions further containa significant amount of oligosaccharides, because thechiro-inositol peak is not completely separated from theoligosaccharide peak as shown in FIG. 2. The chiro-inositolingredients contained in both the pinitol and the chiro-inositolfractions amount to 75-85% of the total chiro-inositol ingredientscontained in the sample soy fraction. In the oligosaccharidefractions harvested at 3.0-4.0 BV, chiro-inositol ingredients arefound at an amount of as low as 0.1-0.5% based on the total solidcontent.

[0045] After passage of the regeneration solution, 3-6 BV ofdistilled water is enough to remove the solvent remaining in theactivated charcoal column, so that the washed activated charcoalcolumn can be used for the next absorption.

[0046] Fourth Process: Concentration of Eluate and Recovery ofSolvent

[0047] Vacuum distillation of each eluate fraction at 60.degree. C.can achieve the concentration as well as the recovery of thesolvent. The vacuum distillation is conducted to a solid content of10% or higher. For use in the next round, the recovered solutioncontaining the solvent is controlled in concentration.

[0048] Fifth Process: Drying

[0049] The concentrate obtained in the fourth process is freeze- orspray-dried to give products as white or yellow powder.

[0050] In accordance with the present invention, theabove-mentioned processes can be modified, as follows, so as toisolate other useful ingredients from soy fractions or improve theproduction efficiency of chiro-inositol ingredients.

[0051] Modification 1: Associated with Recovery of Isoflavone

[0052] In advance of the second process, isoflavone can berecovered from the soy fractions. In this regard, the recovery ofisoflavones resorts to adsorbents (HP resin, Samyang Corp. Korea)(Korean Pat. Laid-Open No. 2000-055133), or to alpha-galactosidasecapable of cleaving isoflavone glycoside bonds (Korean Pat.Laid-Open No. 1998-032766). Where the adsorbents are used,pretreatment effects are also obtained because the adsorbents holda significant amount of proteins thereto with no adsorption ofchiro-inositol ingredients.

[0053] Modification 2: Associated with Recovery of SoybeanOligosaccharides

[0054] The oligosaccharide fractions obtained in the fourth processcontain most of the oligosaccharides present in the soy fraction,as well as being highly free of impurities such as salts orproteins. Thus, soybean saccharide products of high quality can beproduced only by simple purification processes.

[0055] Modification 3: Retreatment of Chiro-Inositol Fractions

[0056] By use of an additional activated charcoal column, thechiro-inositol fractions obtained in the fourth process can bepurified to a higher chiro-inositol content. After being adjustedto the pH range of 3-4, the chiro-inositol fractions are subjectedto activated charcoal column chromatography. At this pH,chiro-inositol is first eluted owing to its weak adsorptive force.The eluate fractions are pooled and can be processed tochiro-inositol products 50% or higher in purity. The retreatmentmay resort to the use of an anion exchange resin (U.S. Pat. No.5,482,631).

[0057] Modification 4: Production of Pinitol Product with HighPurity

[0058] The concentrate of the pinitol fractions obtained in thefourth process is further concentrated to a solid content of 50% ormore and added with an equal volume of acetone at a lowtemperature, after which the solution is allowed to stand for 12hours or more at 10.degree. C. or less to give a precipitate. Thispinitol matter is recovered by centrifugation or vacuum filtrationand dried in vacuo to a purity of 95%.

[0059] Over conventional ion exchange resin process, the activatedcharcoal process of the present invention has the followingadvantages: 1) voluminous samples of low concentrations, such assoybean curd whey, can be treated because chiro-inositolingredients strongly adsorb to activated charcoal and hardly detachtherefrom until they meet specific elution conditions; 2)desalination is not needed in the pretreatment because activatedcharcoal allows salts of the sample to pass without retention; and3) under appropriate elution conditions, high contents ofchiro-inositol ingredients can be obtained in such relatively smallvolumes that the burden of concentrating the eluate, imposed onsubsequent processes, is lightened.

[0060] In order to obtain chiro-inositol with higher purity, theactivated charcoal column chromatography of the present inventionmay be further associated with treatment with microbes prior to thepretreatment process of removing impurities from soy fractions. Inthis regard, for example, soybean curd whey is treated withmicrobes to give a chiro-inositol ingredient-enriched solutionwhich is then deprived of the biomass by centrifugation orfiltration, lo followed by the recovery of chiro-inositol orpinitol of high purity through adsorption to the activated charcoaland other appropriate processes.

[0061] Therefore, in accordance with another aspect of the presentinvention, there is provided a method for recovering chiroinisitolingredients from soy fractions, which comprises a microbialtreatment process of increasing the content of pinitol orchiro-inositol in soy fractions by use of microbes; a pretreatmentprocess of removing the microbial mass generated in the microbialtreatment process, insoluble ingredients, and macromolecules suchas proteins from soy fractions by filtration or centrifugation, anadsorption process of binding matters of interest onto a support byuse of activated charcoal column chromatography or ion-exchangechromatography, an elution process of detaching the matters ofinterest from the support, and a post-treatment process ofrecovering chiro-inositol ingredients as powder.

[0062] FIG. 4 shows a preferred embodiment of this method. First,soy fractions are concentrated and microbes such as bacteria,yeasts, or fungi are cultured in the concentrate to increase thecontent of pinitol or chiro-inositol. Centrifugation or filtrationis conducted to remove the cultured microbes, insoluble matters andpolymeric materials. The filtrate is loaded onto a column filledwith activated charcoal and chiro-inositol ingredients of thefiltrate are adsorbed onto activated charcoal during passagethrough the column. After ingredients remaining unadsorbed arewashed off with distilled water, the adsorbates are eluted withethanol. This eluent is fed at a continuous or stepwiseconcentration gradient from 10 to 50% (v/v). The activated charcoalis reused after washing with distilled water. The eluatescontaining chiro-inositol ingredients are concentrated to desiredpurity.

[0063] In order to produce pinitol with a purity of 90% or more,additional processes are conducted in addition to the basicprocesses of the method. A crystallization process is very usefuland necessary for achieving a pinitol purity of as high as 90%. Toallow the crystallization process, impurities which interrupt thecrystallization of pinitol must be removed, in advance, to theextent that the solution prior to the crystallization has a pinitolpurity of 70% or more. After the microbial treatment, the totalsolids of the soy fractions is analyzed to contain pinitol in anamount of as low as 5-7% with the remainder consisting of proteins,lipids, other carbohydrates, and salts. When this solution isapplied to an activated charcoal column, other ingredients thanpinitol give rise to a decrease in the capacity of the activatedcharcoal and the purity of the pinitol recovered is difficult toincrease to higher than 70%. To avoid this problem, the solutionafter the microbial treatment is concentrated to an extent of atotal solid content of 50-70% (w/w), after which the concentrate isadded with one to three volumes of a 95% ethanol solution. In theresulting solution, all pinitol is dissolved in the supernatantwhile 75% or more of the other ingredients exist as precipitates.After removal of the precipitates by filtration or centrifugation,the supernatant is analyzed to have a pinitol content of 20% ormore. Afterwards the ethanol added can be recovered in asignificant amount by distillation and the remaining ethanol can becompletely removed by a few rounds of distillation with addition ofwater. Then, the pinitol solution deprived of ethanol is loadedonto the activated charcoal column and the adsorbates are elutedwith a 10% ethanol solution to give a pinitol purity of 70% orhigher. As described above, the removal of impurities by solventtreatment prior to loading onto the activated charcoal columnenjoys the advantage of producing pinitol in a high purity,increasing the capacity of the activated charcoal column, andpreventing the lifetime of the activated charcoal from beingreduced owing to the irreversible adsorption of proteins and otherimpurities. Subsequently, the eluate is concentrated to a pinitolconcentration of 600 g chiro-inositol or more, followed bycrystallization in ethanol to produce pinitol in a purity of 90% ormore.

[0064] Therefore, in accordance with a further aspect of thepresent invention, there is provided a method for recoveringpinitol in high purity from soy fractions, which comprises amicrobial treatment process of increasing the content of pinitol orchiro-inositol in soy fractions by use of microbes afterconcentration of the soy fractions; a precipitation process ofaffording the microbial mass generated in the microbial treatmentprocess, insoluble ingredients, and macromolecules such asproteins, as precipitates, a pretreatment process of removing theprecipitates from soy fractions by filtration or centrifugation, anadsorption process of binding molecules of interest onto a supportby use of activated charcoal column chromatography or ion-exchangechromatography, an elution process of detaching the molecules ofinterest from the support, and a post-treatment process ofrecovering chiro-inositol ingredients as powder.

[0065] In FIG. 5, a preferred embodiment of this method isillustrated. First, soy fractions are concentrated and microbessuch as bacteria, yeasts, or fungi are cultured in the concentrateto increase the content of pinitol or chiro-inositol. Thechiro-inositol ingredient-enriched solution is concentrated to atotal solid content of 50-70% (w/w) and added with one to threevolumes of a 95% ethanol solution to give precipitates.Centrifugation or filtration is conducted to remove theprecipitates. From the supernatant or filtrate, ethanol is removedby distillation. Subsequently, the solution free of ethanol ispassed through an activated charcoal column to adsorb pinitol ontothe activated charcoal, followed by elution with a 10% ethanolsolution. The eluate is concentrated, and crystallization inethanol gives pinitol with high purity.

[0066] When associated with other recovery processes, the method ofthe present invention can be used to isolate other usefulingredients, such as isoflavone and soybean oligosaccharides, fromsoy fractions, as illustrated above.

[0067] For use in drugs or foods for the treatment or prophylaxisof diabetes, obesity, or cataracts, the pinitol or chiro-inositolobtained in the present invention is formulated together withpharmaceutically acceptable carriers or added as a usefulingredient to functional beverages or foods.

[0068] A better understanding of the present invention may beobtained in light of the following examples which are set forth toillustrate, but are not to be construed to limit the presentinvention.

EXAMPLE 1

Treatment of Soybean Curd Whey with Saccharomyces calsbergensis

[0069] Saccharomyces calsbergensis was inoculated in sterilesoybean curd whey and after 5 days of culturing, the soybean curdwhey was analyzed for composition change. The results are given inTable 3, below. As seen in Table 3, the total pinitol was increasedby a factor of 2.31 from 0.371 g/L to 0.857 g/L while the totalsugar was reduced by 91%.

3TABLE 3 Composition Change of Soybean Curd Whey after Treatmentwith S. calsbergensis (unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 9.08 20 6.1 6.42 0.528 0.083 0.083 2 11.81 18 2.5 6.420.590 0.080 0.022 3 12.80 15 1.3 6.64 0.648 0.070 0.005 4 12.73 141.2 6.55 0.722 0.080 0.004 5 12.53 14 1.2 6.75 0.857 0.0880.004

EXAMPLE 2

Treatment of Soybean Curd Whey with Saccaromyces cerevisiae

[0070] Saccharomyces cerevisiae was inoculated in sterile soybeancurd whey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 4,below. As seen in Table 4, the total pinitol was increased by afactor of 1.81 from 0.371 g/L to 0.676 g/L while the total sugarwas reduced by 54%.

4TABLE 4 Composition Change of Soybean Curd Whey after Treatmentwith S. cerevisiae (unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 5.81 22 7.0 6.93 0.487 0.093 0.017 2 6.72 20 6.3 7.35 0.0520.100 0.020 3 7.86 20 5.7 8.47 0.554 0.100 0.018 4 7.75 20 5.8 7.810.623 0.111 0.022 5 7.86 19 5.9 7.51 0.676 0.124 0.025

EXAMPLE 3

Treatment of Soybean Curd Whey with Saccaromyces pastorianus

[0071] Saccharomyces pastorianus was inoculated in sterile soybeancurd whey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 5,below. As seen in Table 5, the total pinitol was increased by afactor of 1.59 from 0.371 g/L to 0.590 g/L while the total sugarwas reduced by 60%.

5TABLE 5 Composition Change of Soybean Curd Whey after Treatmentwith S. pastorianus unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 9.20 21 7.1 7.19 0.495 0.090 0.015 2 10.78 20 6.2 6.450.535 0.110 0.017 3 10.68 20 4.6 7.12 0.566 0.122 0.022 4 10.57 205.7 7.07 0.595 0.120 0.023 5 10.53 19 5.2 6.96 0.590 0.1230.023

EXAMPLE 4

Treatment of Soybean Curd Why with Candida utilitis

[0072] Candida utilitis was inoculated in sterile soybean curd wheyand after 5 days of culturing, the soybean curd whey was analyzedfor composition change. The results are given in Table 6, below. Asseen in Table 6, the total pinitol was increased from 0.371 g/,L tothe maximum 0.539 g/L after three days of culturing, and from then,its concentration decreased. The total sugar was reduced by aslittle as 31%.

6TABLE 6 Composition Change of Soybean Curd Whey after Treatmentwith C. utilitis unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 11.01 21 7.3 7.27 0.483 0.105 0.019 2 11.05 20 6.3 7.830.523 0.123 0.023 3 11.12 20 6.2 9.37 0.539 0.124 0.025 4 11.01 206.6 8.67 0.477 0.130 0.007 5 10.82 20 8.9 8.43 0.407 0.1180.008

EXAMPLE 5

Treatment of Soybean Curd Whey with Aspergillus niger

[0073] Aspergillus niger was inoculated in sterile soybean curdwhey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 7,below. As seen in Table 7, the total pinitol was increased from0.371 g/L to the maximum 0.566 g/L after three days of culturing,and since then, decreased to almost zero after five days ofculturing. The total sugar was reduced by as much as 94%.

7TABLE 7 Composition Change of Soybean Curd Whey after Treatmentwith A. niger unit: g/L) C- M- Day OD T. solid T. sugar T. ProteinPinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.134 0.039 10.35 28 13.3 6.12 0.406 0.102 0.056 2 0.15 20 5.7 2.81 0.472 0.1550.023 3 1.30 10 0.7 1.04 0.566 0.176 0.005 4 1.24 8 1.3 1.16 0.2300.052 0.000 5 1.18 8 0.8 1.28 0.060 0.000 0.000

EXAMPLE 6

Treatment of Soybean Curd Whey with Penicillium funiculosum

[0074] Penicillium funiculosum was inoculated in sterile soybeancurd whey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 8,below. As seen in Table 8, the total pinitol was increased from0.371 g/L to the maximum 0.539 g/L after three days of culturing,and from then, its concentration decreased, while the total sugarwas reduced by 49%.

8TABLE 8 Composition Change of Soybean Curd Whey after Treatmentwith P. funiculosum unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 0.93 30 13.9 4.60 0.246 0.082 0.049 2 0.74 30 11.7 4.610.427 0.134 0.087 3 0.75 29 11.9 5.14 0.534 0.192 0.101 4 0.62 269.2 4.92 0.499 0.158 0.063 5 0.99 20 6.6 5.38 0.442 0.144 0.039

EXAMPLE 7

Treatment of Soybean Curd Whey with Trichoderma viride

[0075] Trichoderma viride was inoculated in sterile soybean curdwhey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 9,below. As seen in Table 9, the total pinitol was increased from0.371 g/L to the maximum 0.709 g/L after one day of culturing, andsince then, its concentration decreased, while the total sugar wasreduced by 38%.

9TABLE 9 Composition Change of Soybean Curd Whey after Treatmentwith T. viride unit: g/L) C- M- Day OD T. solid T. sugar T. ProteinPinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.134 0.039 10.8 30 14.2 6.15 0.709 0.237 0.113 2 0.71 29 12.1 5.95 0.578 0.1800.036 3 0.7 29 11.2 6.60 0.518 0.140 0.019 4 0.72 26 10.2 6.100.467 0.172 0.028 5 0.58 24 8.0 5.98 0.352 0.163 0.054

EXAMPLE 8

Treatment of Soybean Curd Whey with Bacillus stearothermophilus

[0076] Bacillus stearothermophilus was inoculated in sterilesoybean curd whey and after 5 days of culturing, the soybean curdwhey was analyzed for composition change. The results are given inTable 10, below. As seen in Table 10, the total pinitol wasincreased by a factor of 1.39 from 0.371 g/L to 0.514 g/L while thetotal sugar was reduced by 29%.

10TABLE 10 Composition Change of Soybean Curd Whey after Treatmentwith B. stearothermophilus (unit: g/L) C- M- Day OD T. solid T.sugar T. Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.620.371 0.134 0.039 1 1.95 26 11.5 7.12 0.539 0.122 0.031 2 2.02 2610.8 7.38 0.477 0.080 0.025 3 2.03 26 9.2 8.22 0.390 0.066 0.020 42.00 26 9.3 7.77 0.438 0.065 0.028 5 3.58 28 9.1 7.98 0.514 0.0690.024

EXAMPLE 9

Treatment of Soybean Curd Whey with Escherichia coli

[0077] Escherichia coli was inoculated in sterile soybean curd wheyand after 5 days of culturing, the soybean curd whey was analyzedfor composition change. The results are given in Table 11, below.As seen in Table 11, the total pinitol was increased by a factor of1.98 from 0.371 g/L to 0.733 g/L while the total sugar was reducedby 31%.

11TABLE 11 Composition Change of Soybean Curd Whey after Treatmentwith E. coli (unit: g/L) C- M- Day OD T. solid T. sugar T. ProteinPinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.134 0.039 13.6 26 11.4 7.79 0.613 0.079 0.043 2 3.59 24 8.8 7.44 0.643 0.0750.045 3 3.50 25 8.7 8.47 0.660 0.070 0.047 4 3.35 24 8.4 7.62 0.7120.082 0.063 5 3.41 24 8.9 8.43 0.733 0.085 0.072

EXAMPLE 10

Treatment of Soybean Curd Whey with Pesudomonas amylodermosa

[0078] Pseudomonas amylodermosa was inoculated in sterile soybeancurd whey and after 5 days of culturing, the soybean curd whey wasanalyzed for composition change. The results are given in Table 11,below. As seen in Table 11, the total pinitol was increased from0.371 g/L to the maximum 0.604 g/L after one day of culturing andsince then, its concenration decreased, while the total sugar wasreduced by as little as 24%.

12TABLE 12 Composition Change of Soybean Curd Whey after Treatmentwith P. amylodermosa (unit: g/L) C- M- Day OD T. solid T. sugar T.Protein Pinitol Inositol Inositol 0 2.00 30 12.9 6.62 0.371 0.1340.039 1 1.81 24 10.8 7.38 0.604 0.085 0.031 2 1.85 24 9.6 7.680.456 0.064 0.025 3 2.30 23 9.8 8.22 0.329 0.040 0.019 4 2.43 239.7 7.33 0.422 0.054 0.025 5 2.36 23 9.8 7.28 0.493 0.061 0.029

EXAMPLE 11

Treatment of Hydrothermal Solution and Suspension of DefattedSoybean with Saccharomyces calsbergensis

[0079] Saccharomyces calsbergensis was inoculated in a hydrothermalsolution and a suspension of defatted soybean and cultured for fivedays. The media were analyzed for composition change during theculturing and the results are given in Table 13, below. After fivedays of culturing, as seen in Table 13, the pinitol content wasincreased by a factor of 2.02 from 0.596 g/L to 1.209 g/L in thehydrothermal solution of defatted soybean, and by a factor of 2.51from 0.618 g/L to 1.551 g/L in the suspension of defattedsoybean.

13TABLE 13 Composition Changes of Hydrothermal Solution andSuspension of Defatted Soybean after Treatment with S.calsbergensis (unit: g/L) Hydrothermal Extract Suspension DayPinitol C-Inositol T. solid T. protein Pinitol C-Inositol T. solidT. Protein 0 0.596 0.114 40 2.55 0.618 0.109 42 2.73 1 0.803 0.10132 2.03 0.817 0.099 34 2.34 2 1.152 0.094 26 2.22 1.230 0.103 322.52 3 1.306 0.103 22 2.35 1.512 0.121 28 2.12 4 1.252 0.108 232.02 1.530 0.118 26 1.99 5 1.209 0.112 24 1.63 1.551 0.115 241.85

EXAMPLE 12

Isolation of Chiro-Inositol Ingredients Using Activated CharcoalColumn

[0080] Experiment 1: Partition Coefficient for Adsorption OntoActivated Charcoal According to pH

[0081] To determine whether activated charcoal was suitable forisolating/L ingredients from other sugars, partition coefficientsof chiro-inositol, pinitol and sugar for adsorption onto activatedcharcoal were examined at various pHs to pH. The results are givenin Table 14, below. Herein, the term "adsorption partitioncoefficient" means the ratio of the concentration of an ingredientadsorbed onto an adsorbent to the concentration of the ingredientremaining unadsorbed under given conditions.

14TABLE 14 Partition coefficients of Chiro-inositol, Pinitol andSugar for Adsorption onto Activated charcoal pH Chiro-inositolPinitol Sugar 3 1.8 1.0 7.5 4 2.0 1.6 7.5 5 4.1 2.2 7.5 6 5.5 2.27.5 7 5.5 2.2 7.8 8 8.4 2.0 7.8 9 4.2 1.6 7.5

[0082] As indicated in Table 14, all chiro-inositol, pinitol andsugars have high partition constants for adsorption onto activatedcharcoal at around neutral pH. Thus, after adsorption is performedunder such pH conditions, elution at acidic pH allowschiro-inositol and pinitol to come out ahead of sugars, therebyseparating the two ingredients from sugars with ease.

[0083] Experiment 2: Composition of Soybean Curd Whey

[0084] The soybean curd whey used in the present invention wasobtained from a soybean curd manufactory in Korea, and analyzed forcomposition. The results are given in Table 15, below.

15TABLE 15 Composition of Soybean Curd Whey Ingredient Content inSolid (%) Content in Sol'n (g/L) Chiro-inositol 0.52 0.13 Pinitol2.04 0.51 Total Sugar 45.0 11.25 Protein 12.0 3.00 Ash 3.0 0.75Others 37.44 9.36 Sum 100.0 25.00

[0085] Experiment 3: Hydrothermal Extraction of Defatted SoybeanMeal

[0086] 200 g of the powder obtained by passing defatted soybeanmeal through a 20 mesh sieve was added with 800 ml of water andextracted at 80.degree. C. for 2 hours with stirring. Thehydrothermal solution was centrifuged at 10,000 rpm to give 600 mlof an aqueous defatted soybean solution (solid content 8.3% (w/v)).This extract was analyzed for composition and the results are givenin Table 16, below.

16TABLE 16 Composition of Hydrothermal Extract of Defatted SoybeanMeal Ingredient Content in Solid (%) Content in Sol'n (g/L)Chiro-inositol 0.44 0.36 Pinitol 1.86 1.54 Total Sugar 39.0 32.4Protein 18.0 14.9 Ash 4.0 3.3 Others 36.7 30.5 Sum 100.0 83.0

[0087] Experiment 4: Composition of Soy Molasses

[0088] Soy molasses obtained from various sources was analyzed forcomposition and the results are given in Table 17, below

17TABLE 17 Composition of Soy Molasses (unit g/100 g) ManufacturerAarhus Central Soya Solbar (Nation) (Dutch) (U.S.A.) (Israel) Brix66.5 60 74 Chiro-inositol 0.4 0.3 0.6 Pinitol 1.5 1.0 1.7 TotalSugar 36.9 28.7 47.0 Protein 9.3 7.6 9.0 Ash 4.0 5.2 6.7

[0089] Experiment 5: Recovery of Chiro-Inositol Ingredients fromSoybean Curd Whey

[0090] 4 liters of soybean curd whey with the composition ofExperiment 2 was filtered to remove insoluble solids, after whichthe filtrate was adjusted to pH 8.0 and loaded at a rate of 500 mlper hour onto a glass column (inner diameter 5 cm.times.length 30cm) packed with 500 ml of activated charcoal. Used was granularactivated charcoal with a size of 30-80 meshes. After completion ofthe adsorption of the filtrate onto activated charcoal, impuritiesremaining unadsorbed were removed by the passage of 500 ml ofdistilled water. Afterwards, flowing with 500 ml of a 10% (v/v)ethanol solution and then with a 50% (v/v) ethanol solution at aflow rate of 500 ml/hr eluted the adsorbates. Thereafter, passageof 2 liters of distilled water through the activated charcoalcolumn made the ethanol remaining therein come out and it wasreused in the next adsorption task. The effluent obtained over theelution period from the start of the elution to the completion ofthe washing was collected in fractions of 100 ml. Each fraction wasanalyzed for contents of pinitol, chiro-inositol and saccharides(sucrose, stachyose, raffinose, fructose and glucose) by HPLC usingDionex Carbonpak MA-1 (Dionex, U.S.A.) as an analysis column withthe aid of a pulsed electrochemical detector, eluting with 69 mMNaOH at a rate of 0.4 ml/min for 90 min. Gas chromatography wasused to analyze the concentration of ethanol in each fraction. InFIG. 2, contents of the compounds in each fraction are shown. Asseen in FIG. 2, pinitol was eluated at 0.6-2.2 BV with a maximumconcentration at 1.6 BV. Chiro-inositol was found mainly in thefractions in the range of 2.0 to 3.2 BV with a maximumconcentration at 2.6 BV. As for oligosaccharides, they emerged wheneluting at 2.4-4.2 BV and their concentration reached at 3.4 BV.The fractions in the range of 2.4-4.2 BV showing pinitol peaks werepooled to give 800 ml which was analyzed to contain pinitol in anamount of 2.03 g/L, chiro-inositol in an amount of 0.11 g/L, andoligosaccharides in an amount of 0.20 g/L. The content of thechiro-inositol ingredients amounted to 61.2% based on the total dryweight of the pinitol fractions. Likewise, the fractions in therange of 2.4-2.8 BV showing chiro-inositol peaks were pooled togive 300 ml which was analyzed to contain pinital in an amount of0.1 g/L, chiro-inositol in an amount of 1.22 g/L, andoligosaccharides in an amount of 4.10 g/L. The content of thechiro-inositol ingredients in the chiro-inositol fraction pool was16% based on the total dry weight of the pool.

[0091] Experiment 6: Recovery of Chiro-Inositol Ingredients fromHydrothermal Extract of Defatted Soybean Meal

[0092] 1.5 liters of a hydrothermal extract of defattedhydrothermal meal having the composition of Experiment 3 wastreated in the same manner as in Experiment 5 to give 900 ml ofpinitol fractions and 300 ml of chiro-inositol fractions whichcontained chiro-inositol ingredients in amounts of 1.92 g/L and1.10 g/L, respectively, based on the total solid weight.

[0093] Experiment 7: Recovery of Chiro-Inositol Ingredients fromSoy Molasses

[0094] 200 g of soy molasses having the composition shown in Table17 of Experiment 4, produced in U.S.A., was diluted with distilledwater to a volume of 1.5 liters. This dilution was treated in thesame manner as in Experiment 5 to give 800 ml of pinitol fractionsand 300 ml of chiro-inositol fractions which containedchiro-inositol ingredients in amounts of 1.88 g/L and 1.40 g/L,respectively, based on the total solid weight.

[0095] Experiment 8: Concentration and Drying of Eluate

[0096] 800 ml of the pinitol pool obtained in Experiment 5 wasconcentrated to a volume of 40 ml in an evaporator maintained at50.degree. C. under vacuum. The concentrate was freeze-dried togive 3.0 g of a pale yellow powder which was analyzed to containchiro-inositol ingredients in an amount of 63.1%. Likewise, 300 mlof the chiro-inositol pool was concentrated to a volume of 30 ml,followed by freeze-drying the concentrate to give 2.4 g of a paleyellow powder. In this powder, chiro-inositol ingredients werefound to amount to 16.5%.

EXAMPLE 13

Recovery of Pinitol at High Efficiency

[0097] 100 L of soybean curd whey with a pinitol content of 0.387g/L was boiled for 20 min to kill autogenous microbes, cooled to30.degree. C., and inoculated with 2 L of precultured Saccharomycescalsbergensis. After incubation for 72 hours with ample supply ofair, centrifugation was conducted to remove the cell mass andinsoluble solid contents. The supernatant, amounting to 95 L, wasmeasured to be increased to 0.793 g/L in pinitol content with nomodulation in chiro-inositol content. The liquid was passed at arate of 20 L/hour through a column packed with 10 L of activatedcharcoal to adsorb pinitol onto the activated charcoal. Followingwashing the activated charcoal with 10 L of distilled water,elution of pinitol was carried out with 10 L of 10% ethanol at aflow rate of 10 L/hr. Afterwards, 10 L of 50% ethanol was flowed ata rate of 10 L/hr into the activated charcoal column to removesaccharides therefrom. For use in the next adsorption, theactivated charcoal was washed with distilled water.

[0098] From the start of eluent feeding, the eluates were collectedin fraction by 2 L. Each fraction was analyzed for contents ofpinitol and total saccharides and the results are given in FIG. 3.The fractions showing a pinitol peak were pooled to 14 L which wasthen freeze-dried to give 90.2 g of a pale yellow powder with apinitol purity of 68.0%. Chiro-inositol fractions were notrecovered owing to low chiro-inositol contents.

EXAMPLE 14

Production of Pinitol of High Purity

[0099] 1,000 L of soybean curd whey with a pinitol concentration of0.35 g/L was 10-fold concentrated to 100 L. The concentrate wascooled to 30.degree. C. and inoculated with Saccharomycescalsbergensis in the same manner as in Example 13 in a 150 L tank.The yeast was incubated for 48 hours with sufficient aeration,followed by centrifugation to remove the cell mass and floatingmatters. The supernatant, amounting to 95.5 L, was analyzed tocontain a pinitol content of 7.78 g/L, and 5-fold concentrated to19.1 L. Adding 30 L of a 95% ethanol solution to the concentrateled to removal of 76.5% of the solid content present in theconcentrate while the purity of pinitol reached 25% with a 4-foldincrease. From the dilution, the ethanol was almost completelyremoved by repeating the addition and evaporation of water. 20 L ofthe ethanol-removed solution was loaded onto a 20 L activatedcharcoal column in the same manner as in Example 13 to adsorbpinitol onto the activated charcoal. Following washing with 20 L ofdistilled water, pinitol was eluted with 20 L of a 10% ethanolsolution. The eluate was 20-fold concentrated to 1 L, added with1.5 L of a 95% ethanol solution, and allowed to stand for 12 hoursat room 5 temperature with slow stirring to give pinitol as aprecipitate. It was recovered by vacuum filtration and dried at40.degree. C. in vacuo to give 495 g of a white powder with apinitol purity of 96.5%. The material balance of the whole processis summarized in Table 18.

18TABLE 18 Material Balance of the Process for High Purity PinitolProduction Process Vol. (L) Conc. (g/L) T. Solid (g) PI Content(g/L) PI (g) PI Purity (%) Recovery Effic. (%) Whey 1000 18 18,0000.35 350 1.9 Concentrated 100 178 17,800 3.49 349 2.0 WheyMicrobial 95.5 126 12,033 7.78 743 6.2 100 Treatment &Centrifugation Concentration 19.1 630 12,020 38.9 742 6.2 99.9Solvent Add 49.1 57.6 2,828 14.1 691 25.0 93.0 Adsorption &20.0 45.0 832 31.3 626 75.2 84.3 Elution Concentration 2.5 359.6832 250.0 625 75.1 84.1 Crystallization 495 g 99.0% 490 96.5% 47897.5% 64.3 & Drying

INDUSTRIAL APPLICABILITY

[0100] As described hereinbefore, the method of the presentinvention can convert pinitol derivatives of soy fractions topinitol by microbial treatment, thereby recovering pinitol atmaximum efficiency. Additionally, other saccharides can be removedfrom the soy fractions, thereby brining about a significantreduction in the burden of subsequent recovery processes as well asin the load of organics contained in the final waste. Further, theactivated charcoal column chromatography of the present inventionenables the treatment of more voluminous samples of lowconcentrations, compared to ion exchange chromatography. Anotheradvantage of the present invention is that desalination is notneeded in the pretreatment because activated charcoal allows saltsof the sample to pass without retention. The present invention alsoenjoys the advantage that, under appropriate elution conditions,high contents of chiro-inositol ingredients can be obtained in suchrelatively small volumes that the burden of concentrating theeluate, imposed on subsequent processes, is lightened.Consequently, the novel activated charcoal column chromatographyassociated with the microbial treatment can isolate chiro-inositolingredients from soy fractions at far higher efficiency than canprior arts.

[0101] The present invention has been described in an illustrativemanner, and it is to be understood that the terminology used isintended to be in the nature of description rather than oflimitation. Many modifications and variations of the presentinvention are possible in light of the above teachings. Therefore,it is to be understood that within the scope of the appendedclaims, the invention may be practiced otherwise than asspecifically described.

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