Lentinan extraction process from mushrooms using ionic liquid

Abstract

A method of extracting lentinan from lentinan containing mushrooms comprising the step of contacting the mushrooms with an ionic liquid so as to obtain a lentinan rich ionic liquid solution.

Claims

1. A method of extracting lentinan from lentinan containing mushrooms comprising a step of contacting the mushrooms with an ionic liquid in order to obtain a lentinan rich ionic liquid solution and wherein the contact time is between 10 minutes and 48 hours and the contacting takes place at a temperature of 15 to 90 C., further comprising a step of pre-treating the lentinan containing mushrooms at a temperature of less than 100 C.

2. The method of claim 1, wherein the ionic liquid is hydrophilic.

3. The method of claim 2, wherein the ionic liquid has at least one member of a group consisting of a hydrophilic cation and a hydrophilic anion.

4. A method of claim 3, wherein the cation is selected from ammonium, azaannulenium, azathiazolium, benzofuranium, borolium, diazabicyclodecenium, diazabicyclononenium, diazabicycloundecenium, dithiazolium, furanium, imidazolium, indolinium, indolium, morpholinium, oxaborolium, oxaphospholium, oxazinium, oxazolium, iso-oxazolium, oxothiazolium, pentazolium, phospholium, phosphonium phthalazinium, piperaziniumpiperidinium, pyranium, pyrazinium, pyrazolium, pyridazinium, pyridinium, pyrimidinium, pyrrolidinium, pyrrolium, quinazolinium, quinolinium, iso-quinolinium, quinoxalinium, selenozolium, tetrazolium, iso-thiadiazolium, thiazinium, thiazolium, thiophenium, triazadecenium, triazolium and iso-triazolium.

5. The method of claim 3, wherein the cation is selected from: ##STR00009## wherein: R.sup.a, R.sup.b, R.sup.c, R.sup.d, R.sup.e, R.sup.f and R.sup.g are each independently selected from hydrogen, a C.sub.1 to C.sub.10, straight chain or branched alkyl group, a C.sub.3 to C.sub.8 cycloalkyl group, or a C.sub.6 to C.sub.10 aryl group, or any two of R.sup.b, R.sup.c, R.sup.d, R.sup.e and R.sup.f attached to adjacent carbon atoms form a methylene chain (CH.sub.2).sub.q wherein q is from 3 to 6; and wherein said alkyl, cycloalkyl or aryl groups or said methylene chain are unsubstituted or may be substituted by one to three groups selected from: C.sub.1 to C.sub.6 alkoxy, C.sub.3 to C.sub.8 cycloalkyl, C.sub.6 to C.sub.10 aryl, C.sub.7 to C.sub.10 alkaryl, C.sub.7 to C.sub.10 aralkyl, O, OH, NO.sub.2, CO.sub.2R.sup.x, OC(O)R.sup.x, C(O)R.sup.x, NR.sup.xC(O)NR.sup.yR.sup.z, NR.sup.xC(O)OR.sup.y, OC(O)NR.sup.yR.sup.z, C(O)NR.sup.yR.sup.z, NR.sup.yR.sup.z, or a heterocyclic group, wherein R.sup.x, R.sup.y and R.sup.z are independently selected from hydrogen or C.sub.1 to C.sub.6 alkyl.

6. The method of claim 5, wherein R.sup.a, R.sup.b, R.sup.c, R.sup.d, R.sup.e, R.sup.f and R.sup.g are each independently selected from hydrogen, a C.sub.1 to C.sub.10, straight chain or branched alkyl.

7. The method of claim 5, wherein R.sup.a and R.sup.g are each independently selected from C.sub.1 to C.sub.8, linear or branched, alkyl.

8. The method of claim 5, wherein R.sup.a and R.sup.g are each independently selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl.

9. The method of claim 5, wherein R.sup.b, R.sup.c, R.sup.d, R.sup.e and R.sup.f, are each hydrogen or are each independently selected from C.sub.1 to C.sub.8, linear or branched, alkyl.

10. The method of claim 5, wherein R.sup.b, R.sup.c, R.sup.d, R.sup.e and R.sup.f are each hydrogen or are each independently selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl.

11. The method of claim 5, wherein R.sup.b, R.sup.c, R.sup.d, R.sup.e and R.sup.f are each hydrogen.

12. The method of claim 5, wherein the cation is selected from: ##STR00010## wherein: R.sup.a, R.sup.b, R.sup.c, R.sup.d, R.sup.e, R.sup.f, and R.sup.g are defined in claim 5.

13. The method of claim 12, wherein the cation is selected from: ##STR00011## wherein: R.sup.a and R.sup.g are as defined in claim 5, and R.sup.h represents hydrogen or a methyl group.

14. The method of claim 5, wherein the cation is selected from: ##STR00012##

15. A method of claim 3, wherein the cation is selected from:
[N(R.sup.a)(R.sup.b)(R.sup.c)(R.sup.d)].sup.+ wherein: R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently selected from hydrogen, a C.sub.1 to C.sub.10, straight chain or branched alkyl group, a C.sub.3 to C.sub.8 cycloalkyl group, or a C.sub.6 to C.sub.10 aryl group; and wherein said alkyl, cycloalkyl or aryl groups or said methylene chain are unsubstituted or may be substituted by one to three groups selected one to three groups selected from: C.sub.1 to C.sub.6 alkoxy, C.sub.3 to C.sub.8 cycloalkyl, C.sub.6 to C.sub.10 aryl, C.sub.7 to C.sub.10 alkaryl, C.sub.7 to C.sub.10 aralkyl, O, OH, NO.sub.2, CO.sub.2R.sup.x, OC(O)R.sup.x, C(O)R.sup.x, NR.sup.xC(O)NR.sup.yR.sup.z, NR.sup.xC(O)OR.sup.y, OC(O)NR.sup.yR.sup.z, C(O)NR.sup.yR.sup.z, NR.sup.yR.sup.z, or a heterocyclic group, wherein R.sup.x, R.sup.y and R.sup.z are independently selected from hydrogen or C.sub.1 to C.sub.6 alkyl, and wherein one of R.sup.a, R.sup.b, R.sup.c and R.sup.d may also be hydrogen.

16. The method of claim 15, wherein R.sup.a, R.sup.b, R.sup.c and R.sup.d are each independently selected from hydrogen, a C.sub.1 to C.sub.10, straight chain or branched alkyl.

17. The method of claim 15, wherein one of R.sup.a, R.sup.b, R.sup.c and R.sup.d is hydrogen.

18. The method of claim 15, wherein R.sup.a, R.sup.b, R.sup.c and R.sup.d are independently selected from methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl.

19. The method of claim 15, wherein two or more of R.sup.a, R.sup.b, R.sup.c and R.sup.d are independently selected from methyl, ethyl, propyl and butyl.

20. The method of claim 15, wherein three or more of R.sup.a, R.sup.b, R.sup.c and R.sup.d are independently selected from methyl, ethyl, propyl and butyl.

21. The method of claim 15, wherein R.sup.b, R.sup.c, and R.sup.d are each the same alkyl group selected from methyl, ethyl n-butyl, and n-octyl, and R.sup.a is selected from hydrogen, methyl, n-butyl, n-propyl, n-pentyl, n-hexyl, n-hepty n-octyl, n-tetradecyl, 2-hydroxyethyl, or 4-hydroxy-n-butyl.

22. The method of claim 15, wherein the cation is selected from: ##STR00013##

23. The method of claim 1, wherein the anion is selected from inorganic anions, sulfonate anions, fluorinated anions, sulphate anions, carboxylate anions, carbonate anions and metal anions.

24. The method of claim 23, wherein the anion is selected from [F].sup., [Cl].sup., [Br].sup., [CH.sub.3CO.sub.2].sup. and [CH.sub.3CH.sub.2CO.sub.2].sup..

25. The method of claim 23, wherein the inorganic anions are selected from [F].sup., [Cl].sup., [Br].sup., [I].sup., [NO.sub.3].sup., [BF.sub.4], [HSO.sub.4].sup., and [SO.sub.4].sup.2; the fluorinated anions are selected from [CF.sub.3CO.sub.2].sup., [CF.sub.3SO.sub.3].sup. and [(C.sub.2F.sub.5).sub.3PF.sub.3].sup.; the sulfonate anions are selected from [CH.sub.3SO.sub.3].sup., [C.sub.2H.sub.5SO.sub.3].sup. and [bis(2-ethylhexyl)-sulfosuccinate].sup. (also referred to as [AOT].sup.); the carboxylate anions are selected from [CH.sub.3CO.sub.2].sup. and [CH.sub.3CH.sub.2CO.sub.2].sup.; the carbonate anion is [HCO.sub.3].sup.; or the sulphate anions are selected from [CH.sub.3OSO.sub.3].sup., [C.sub.2H.sub.5OSO.sub.3].sup., [C.sub.8H.sub.17OSO.sub.3].sup., and [H.sub.3C(OCH.sub.2CH.sub.2).sub.nOSO.sub.3].sup..

26. The method of claim 1, wherein the ionic liquid further comprises water, acetate, lactates, dicyanamides, aqueous phosphates or urea.

27. The method of claim 26, wherein the ionic liquid includes water, and wherein the non-water ionic liquid component to water ratio is from 99:1 to 50:50.

28. The method of claim 26, wherein the ionic liquid includes water, and wherein the non-water ionic liquid component to water ratio is from 99:1 to 75:25.

29. The method of claim 1, wherein the contacting takes place at a temperature of 15 to 80 C.

30. The method of claim 1, wherein the contacting takes place at a temperature of 60 to 80 C.

31. The method of claim 1, wherein the contact time is from 30 minutes to 4 hours.

32. The method of claim 1, wherein the lentinan containing mushrooms are in wet form are masticated prior to contacting with the ionic liquid.

33. The method of claim 1, further comprising a step of drying the lentinan rich ionic liquid solution.

34. The method of claim 1, further having a step from at least one member of a group consisting of: filtering the lentinan rich ionic liquid solution to remove mushroom matter; precipitating the lentinan from the lentinan rich ionic liquid solution; washing the precipitated lentinan; drying the precipitated lentinan; freeze drying the precipitated lentinan; spray drying the precipitated lentinan; purifying the lentinan using counter-current chromatography; and subjecting extracted lentinan to a dialysis process.

35. The method of claim 34, wherein an alcohol is used for any precipitation.

36. The method of claim 35, wherein the alcohol is selected from methanol, ethanol, iso-propanol and combinations thereof.

37. The method of claim 34, wherein an ionic liquid based counter-current chromatography solvent system is used for any counter-current chromatography.

38. The method of claim 1, wherein the pre-treatment step comprises a wax removal step.

39. The method of claim 1, wherein the pre-treatment step comprises a steroid removal step.

40. The method of claim 1, wherein the lentinan containing mushrooms have not been dried prior to contacting with the ionic liquid.

41. The method of claim 1, wherein the pre-treatment step comprises a sugar removal step.

42. A method of extracting lentinan from lentinan containing mushrooms comprising a step of contacting the mushrooms with an ionic liquid in order to obtain a lentinan rich ionic liquid solution and wherein the contacting takes place at a temperature of 15 to 90 C., further comprising a step of pre-treating the lentinan containing mushrooms at a temperature of less than 100 C., and wherein the pre-treatment step comprises a wax removal step.

43. A method of extracting lentinan from lentinan containing mushrooms comprising a step of contacting the mushrooms with an ionic liquid in order to obtain a lentinan rich ionic liquid solution and wherein the contacting takes place at a temperature of 15 to 90 C., further comprising a step of pre-treating the lentinan containing mushrooms at a temperature of less than 100 C., and wherein the pre-treatment step comprises a steroid removal step.

Description

(1) The present invention will now be described by way of example and with reference to the accompanying figures in which:

(2) FIG. 1 is a gel-HPLC analysis of lentinan extracted from mushrooms using [C.sub.4mim]Cl;

(3) FIG. 2 is a gel-HPLC analysis of the lentinan extraction shown in FIG. 1, wherein the extract has been maintained at 100 C. for six days;

(4) FIG. 3 is a gel-HPLC analysis of the commercial lentinan extracts as supplied;

(5) FIG. 4 is a gel-HPLC analysis of commercial lentinan extracts, purified with [C.sub.4mim]Cl;

(6) FIG. 5 is a further gel-HPLC analysis of commercial lentinan extracts, purified with [C.sub.4mim]Cl; and

(7) FIG. 6 is a stationary phase retention curve for a CCC solvent system at 30 C. and 40 C.

GENERAL PROCEDURE FOR LENTINAN FROM DRIED SHITAKE MUSHROOM GRANULES

Example 1

(8) 5 Kg of Shitake mushrooms were dried and ground up to form small granules. This mushroom dry matter was purified to lentinan in a 3 step process, wherein fatty acids and other oils are first removed using ethyl ethanoate, followed by the removal of mono-saccharides using ethanol and finally a lentinan extraction using an ionic liquid.

(9) The extraction involved heating the mushroom granules (250 g) with ethyl ethanoate for 4 to 6 hours under reflux, followed by filtration of the mushroom powder. The extraction was carried out twice with ethyl ethanoate and twice with boiling industrial methylated spirits (IMS).

(10) The ethyl ethanoate extracts contained fatty acid derived compounds and other oils such as steroids, for example ergosterol. The ethanol extract removed mono-saccharides such as mannitol.

(11) Ergosterol was easily isolated in pure form by suspending the ethyl ethanoate extract in water/hexane leading to the precipitation of ergosterol.

(12) The ethyl ethanoate and IMS solvents were recovered on a rotary evaporator and reused. It will be appreciated that such a step saves on overall solvent usage and reduces the cost of the overall process.

(13) The resulting brown granules were then extracted with ionic liquid (1000 g), using [C.sub.4mim]Cl, for 4 to 48 hours at 70 to 90 C. The ionic liquid extract was mixed with cold water (10 to 30 wt %) and vacuum filtered through a polypropylene filter. Centrifuging at 4200 rpm for 10 to 15 minutes can also be used.

(14) The supernatant was evaporated under vacuum to remove water (80 C. maximum temperature) then mixed gradually with an equal volume of IMS. The precipitate that formed was isolated by centrifugation (4200 rpm). The resulting solution was mixed with 3 volumes of IMS and the resultant precipitate was isolated using a centrifuge under the same conditions.

(15) The precipitate obtained was washed with IMS until a clear supernatant was observed. The grey-brown precipitate obtained contained lentinan, and was subsequently freeze-dried to a light brown powder (approximately 15 g), with a glycans type polysaccharide content of 10 to 70%, by gel HPLC. This is referred to as crude lentinan.

(16) The solid cake that resulted after the first centrifugation from ionic liquid was re-extracted with ionic liquid under similar conditions and was subject to the same precipitation procedure using IMS, to give a second sample of crude lentinan. The amount of crude lentinan was considerably reduced.

(17) The FIG. 1 chromatogram shows three peaks at approximately 9.5, 10 and 13 minutes corresponding to the triple-helix conformation, the random coil conformation and mono-saccharides respectively. The chromatogram demonstrates that a large portion of the lentinan extracted is maintained in the triple helix structure. The ratio of triple helix to random coil conformation is approximately 60:40.

(18) FIG. 2 demonstrates that effect of heating the extract to 100 C. for six days. The chromatogram shows the presence of three peaks at 9, 10.5 and 13 minutes corresponding to the triple-helix conformation, the random coil conformation and mono-saccharides respectively. As discussed above, there is a loss in triple-helix conformation when lentinan is exposed to high temperatures for sustained periods of time. FIG. 2 clearly shows a decrease in the proportion of triple-helix and random coil conformations compared to FIG. 1, indicating that the lentinan has been denatured.

Example 2

(19) The above process was repeated with choline chloride as the ionic liquid extractant for the mushroom granules.

(20) Directly after the extraction with ethyl acetate and ethanol, the mushroom granules were quickly washed with water and filtered. As choline chloride has a melting point of 300 C., it must be used with small amounts of water added to it (in this case, a 2:1 ratio of choline chloride to water is used) to obtain an ionic liquid.

(21) After heating the mushroom granules with this wet choline chloride for 30 minutes, the mixture was filtered through a polypropylene filter and was washed with hot water on the filter 3 times. The liquid extracts were combined and the water was evaporated to give a choline chloride solution of crude lentinan. This was then treated as before in the IMS precipitation stages.

Example 3

(22) 300 g of dried Shitake mushrooms were washed with 2 liters of boiling industrial methylated spirits (IMS) and then filtered to recover the IMS.

(23) The resulting precipitate was washed with water and then filtered, with the water phase being disposed.

(24) The resulting precipitate was then washed with approximately 3 to 5 liters of hot water (temperature of from 80 C. to less than 100 C. This wash step was repeated three times. Following washing, the water was removed by means of a rotary evaporator to give a crude lentinan gel.

(25) The gel was dissolved in a mixture of [C.sub.4mim]Cl (1-butyl-3-methylimidazolium chloride) (50 g) and water (50 ml), with any remaining solids being filtered off.

(26) The solution was then carefully added to IMS to precipitate out the lentinan, which was then dried and ground to a powder.

(27) A yield of 1.5 g was obtained.

(28) Extraction of Lentinan from Commercial Water Extractions of Shitake Mushrooms.

(29) For low MW lentinan, derived from Chinese freeze dried mushroom extracts, labelled and assayed 50% polysaccharides, the water soluble lentinan polysaccharides contained therein can be isolated in over 90% purity as measured on a gel-HPLC column.

Example 4

(30) The lentinan extract comes as a yellow-brown powder, which is mostly soluble in water.

(31) The powder (5.0 g) was completely dissolved in a liquid mixture of 80 to 90% [C.sub.4mim]Cl and 10 to 20% water mixture at room temperature or up to 40 C. Industrial alcohol (97 to 98% ethanol and 2 to 3% methanol mixture) (5 ml) was carefully added to a stirred solution of the crude mushroom extract in [C.sub.4mim]Cl.

(32) During the addition, an off-white precipitate formed that was isolated by vacuum filtration using a 30 ml S2 sintered filtered funnel. The precipitate was washed with a 50/50 mixture of industrial alcohol and water, to give an off-white powder on drying (1.7 g).

(33) Analysis of the off white powder revealed that its 1H NMR (low resolution in D.sub.2O) was consistent with literature spectra, and the gel-HPLC analysis showed that it was a single substance with a retention time of 8 minutes (see FIGS. 3 and 4).

(34) FIG. 3 relates to the untreated extract (Note, the peaks around 15 minutes are an artefact of the HPLC process and are not due to the presence a compound). Three elution peaks can be observed at approximately 8.5, 9.5 and 10.5 minutes corresponding to the triple-helix conformation, the inactive random coil conformation and the presence of mono-saccharides respectively. The chromatogram indicates a large proportion of the extract correspond to the mono-saccharides and other non-polymeric compounds.

(35) FIG. 4 relates to the treated extract with an estimated purity of 90 to 95% lentinan (Note, the peaks around 15 minutes in FIGS. 3 and 4 are believed to be an artefact of the HPLC process and are not due to the presence of a second compound). After purification of the lentinan extract using [C.sub.4mim]Cl only one elution peak is observed between 8 and 9 minutes. The results indicate that the ionic liquids are, at least partially, selective towards to the active triple-helix structure.

Example 5

(36) The yellow-brown powder (350 g) was completely dissolved in a liquid mixture of 50-50 [C.sub.4mim]Cl and water (700 ml) at 40 C. Industrial alcohol (97 to 98% ethanol and 2 to 3% methanol mixture) (700 ml) was carefully added to a stirred solution of the crude mushroom extract in [C.sub.4mim]Cl.

(37) During the addition, a pale brown precipitate formed that was isolated by vacuum filtration using a 1 liter S1 sintered filtered funnel. The precipitate was washed with a 50/50 mixture of industrial alcohol and water, to give an off pale brown powder on drying. The precipitate was dissolved in water (250 ml), and was re-precipitated from water with industrial alcohol (250 ml). This stage gave only marginal improvements in purity and demonstrates that the ionic liquid is essential in the purification process.

(38) Finally, the light brown precipitate was dissolved in [C.sub.4mim]Cl/water (80/20), and precipitated with industrial alcohol, to give a white powder.

(39) The analysis of the off white powder revealed that its 1H NMR (low resolution in D.sub.2O) was consistent with literature spectra, and the gel-HPLC analysis showed that it was over 95% lentinan, with a retention time of 8 minutes (see FIG. 5). The estimated purity is 90 to 95% lentinan.

(40) For reference, the peaks around 15 minutes shown in FIG. 5 are believed to be an artefact of the HPLC process and are not due to the presence of other compounds. The small peak at 7 minutes is a high molecular weight impurity and accounts for only 4% of the product.

(41) FIG. 5 relates to the purification of commercially available lentinan (FIG. 3), wherein the extraction solution comprises a 50:50 water/[C.sub.4mim]Cl solution. The chromatogram only displays one elution peak between 7 and 9.5 minutes. The purification method using a 50:50 water/[C.sub.4mim]Cl solution produces a much broader peak than that observed when using a solution mixture of 80 to 90% [C.sub.4mim]Cl and 10 to 20% water mixture (FIG. 4). The broader resonance corresponds to an increase in the range of molecular weight of the lentinan extracted.

(42) Purification of Lentinan Extracts Using Counter-Current Chromatography (CCC)

(43) The Chinese freeze dried mushroom extracts (yellow-brown powder) were further purified using counter-current chromatography (CCC).

(44) In order to determine the required flow rate, the retention time of the stationary was first assessed (see FIG. 6).

(45) A dipotassium phosphate solution in water (250 ml) was mixed with [C.sub.4mim]Cl (250 ml) and industrial methylated spirits (50 ml). Degassing of the solution was achieved using sonification, after which two phases were formed, an aqueous phase (stationary phase) and a less dense ionic liquid phase (mobile phase).

(46) The retention experiments were performed using an IL-prep CCC, based on a J-type centrifuge containing a 133 ml, 2.1 diameter coil of 36 meters in length. Both phases were pumped into the column wherein the pump for the stationary phase was located at the bottom of the reservoir and the pump for the mobile phase located at the top of the reservoir. The experiments were performed using a rotation speed of 865 rpm and at temperatures of 30 C. and 40 C.

(47) FIG. 6 illustrates the dependency of stationary phase retention on the flow rate of the mobile phase. FIG. 6 indicates that the highest level of retention is achieved at conditions of 1 to 2 ml/min at 40 C., 865 rpm.

Example 6

(48) The mobile phase was pumped into a CCC column, containing only stationary phase, at a low rate of 2.5 ml/min. Once phase retention became constant at 67%, the flow rate of the mobile phase was reduced to 1.0 ml/min.

(49) Commercial lentinan (1.5 g) was dissolved in a mixture of ionic liquid mobile phase (2.5 ml) and aqueous stationary phase (5 ml). Once dissolved, the solution was loaded on to the 5.0 ml injection loop and subsequently injected onto the 133 ml coil at a flow rate of 1.0 ml/min.

(50) A total of 40 fractions were collected, each fraction containing 5 ml of mobile phase. Fractions 23 to 35 were combined and ethanol was added to the solution in order to precipitate lentinan. The precipitate was subsequently filtered, washed using ethanol, and dried to produce a slightly off-white powder (0.45 g).

(51) It was noted that a small amount of stationary phase bleeding occurred preceding the elution of lentinan.